CN108333013A - A kind of preparation method of jujube flower paraffin section - Google Patents
A kind of preparation method of jujube flower paraffin section Download PDFInfo
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- CN108333013A CN108333013A CN201810137942.2A CN201810137942A CN108333013A CN 108333013 A CN108333013 A CN 108333013A CN 201810137942 A CN201810137942 A CN 201810137942A CN 108333013 A CN108333013 A CN 108333013A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 24
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- 238000012545 processing Methods 0.000 claims abstract description 14
- 241001247821 Ziziphus Species 0.000 claims abstract 55
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 195
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 72
- 239000011521 glass Substances 0.000 claims description 52
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 40
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- 238000006297 dehydration reaction Methods 0.000 claims description 21
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 20
- 235000019441 ethanol Nutrition 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 16
- 230000007935 neutral effect Effects 0.000 claims description 14
- 239000012153 distilled water Substances 0.000 claims description 13
- 229960000583 acetic acid Drugs 0.000 claims description 10
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- 238000002791 soaking Methods 0.000 claims description 10
- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical compound OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 claims description 9
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- QUBBAXISAHIDNM-UHFFFAOYSA-N ethyldimethylbenzene Natural products CCC1=CC=CC(C)=C1C QUBBAXISAHIDNM-UHFFFAOYSA-N 0.000 claims description 6
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- 208000005156 Dehydration Diseases 0.000 description 19
- 235000008529 Ziziphus vulgaris Nutrition 0.000 description 17
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 10
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 9
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
Abstract
The present invention provides a kind of preparation methods of jujube flower paraffin section, belong to paraffin section technical field.The preparation method includes the following steps：1) jujube flower bud is fixed；The jujube flower bud is with the stem section that a segment length is 0.3~0.5cm；2) it is dehydrated；3) the first transparent processing；4) the second transparent processing；5) waxdip；6) it embeds；7) wax is repaiied；8) it is sliced；9) piece is opened up；10) it dewaxes；11) rehydration；12) secondary rehydration；13) it dyes；14) cover plate, mounting.Jujube flower paraffin section preparation method provided by the invention, it can obtain the complete longitudinal section of blastular, and then facilitate observation blastular inside cell and institutional framework development change and jujube flower cellular morphology, while by observe jujube flower paraffin section can obtain Embryo Sac Development structure variation and reproductive process in basic law.
Description
Technical field
The invention belongs to paraffin section technical fields, and in particular to a kind of preparation method of jujube flower paraffin section.
Background technology
Jujube tree (Ziziphusjujuba Mill.) is native to China, is a kind of important economic tree.Its fruit shape beauty,
Delicious flavour, rich in benefit materials such as a variety of amino acid of needed by human body, vitamin, polysaccharide, have very high food therapy value and
Health-care efficacy, it is deep to be liked by consumers in general.Crossbreeding is one of most important method of current fruit tree of new species cultivation, but
Jujube tree crossbreeding fluctuates for a long time, and main cause is that jujube tree is hermaphrodite flower, and flower very little causes emasculation difficult, and falls
Spend shedding serious, the low equal biological characteristics of fruit-setting rate are unfavorable for crossbreeding and seed selection, while at present in terms of jujube tree embryology
Relevant research is not deep enough, also limits the development of jujube tree genetic improvement work to a certain extent so that jujube tree germplasm innovation
More fall behind.
Paraffin section is the important experimental technique in Histological research, and basic step includes materials and fixes, is dehydrated, thoroughly
Bright, waxdip embeds, repaiies wax, slice, exhibition piece and dry a series of specially treateds such as piece, dewaxing, rehydration, dyeing, mounting and observation.
Although whole operation process is uncomplicated, the slice of high quality is made, the operation of each step is critically important, and has
Processing method needed for the plant of different tissues structure and the institutional framework of plant different parts is different.
Currently, the research for carrying out paraffin section technology using jujube flower as material is less, and dicing effect is not especially desirable, master
It is that jujube flower is smaller and younger tender to want problem, be easy to cause slice it is broken it is imperfect, institutional framework is unintelligible, be unfavorable for observing.
It is more weak in view of current jujube tree biology of reproduction Research foundation, and paraffin section technology again be research plant biological property compared with
Good method, so the preparation method of suitable jujube tree flower paraffin section is found, to embryo sac tissue in observation jujube flower growth course
The variation of structure and the basic law of reproductive process are of great significance.
Invention content
In view of this, the purpose of the present invention is to provide a kind of preparation method of jujube flower paraffin section, prepared by the method
Biopsy tissues structural integrity clearly be easy to observe.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
The present invention provides a kind of preparation methods of jujube flower paraffin section, include the following steps：
1) jujube flower bud is placed in fixer, fixes 24~36h, obtain fixed jujube flower；
The jujube flower bud is with the stem section that length is 0.3~0.5cm；
The fixer be 50% by formalin, glacial acetic acid and volumetric concentration that volumetric concentration is 38% ethyl alcohol
Aqueous solution is 0.95~1.05 by volume:0.95~1.05:17.5~18.5 configurations obtain；
2) the fixation jujube flower in the step 1) is dehydrated, obtains dehydration jujube flower；
3) the dehydration jujube flower in the step 2) is mixed with the first clarifier to immersion and carries out the first transparent processing, obtains the
One transparent jujube flower；
It according to volume ratio is 0.95~1.05 that first clarifier, which is absolute ethyl alcohol and dimethylbenzene,：0.95~1.05 mixing
The mixed liquor of formation；
4) the first transparent jujube flower in the step 3) with dimethylbenzene is mixed to immersion and carries out the second transparent processing, obtain second
Transparent jujube flower and dimethylbenzene system；
5) it is 54~57 DEG C of atoleines, shape to sequentially add dimethylbenzene and temperature to the second transparent jujube flower of the step 4)
At the dimethylbenzene paraffin mixed liquor containing the second transparent jujube flower 35~37h is toasted at 39~41 DEG C, then with every 1.4~1.6h
The rate of 3 DEG C of heating is warming up to 57~59 DEG C of 2.8~3.2h of heat preservation, forms pre- waxdip jujube flower；With 57~59 DEG C after the heat preservation
Atoleine replace the pre- waxdip jujube flower of dimethylbenzene paraffin mixed liquid dipping, obtain waxdip jujube flower；
6) by the step 5) waxdip jujube flower and 57~59 DEG C of atoleine pour into embedded box and wrap simultaneously
It buries, the position of waxdip jujube flower is adjusted after 2~3s, keeps waxdip jujube flower parallel with embedded box bottom surface, it is cooling, obtain embedding jujube flower wax
Block；
7) it carries out the embedding jujube flower wax stone in the step 6) to repair wax, obtains finishing wax stone；
8) the finishing wax stone in the step 7) is sliced, obtains jujube flower slice wax band；
The thickness of the jujube flower slice wax band is 7.5~8.5 μm；
9) the jujube flower slice wax band that the step 8) obtains is transferred in 23~28 DEG C of water, jujube flower slice wax band is existed
Unfold in water, the jujube flower slice wax band after unfolding is transferred on glass slide, and the jujube flower being supported on glass slide is sliced wax band
Piece is opened up, obtains being supported on the jujube flower slice on glass slide；
10) by the jujube flower slice xylene soak dewaxing 13 of being supported on glass slide of obtaining of the step 9)~
17min replaces dimethylbenzene and impregnates 10~15min of dewaxing again, the jujube flower slice to be dewaxed；
11) 9~11min of rehydration is impregnated in the jujube flower slice rehydration agent for the dewaxing for obtaining the step 10), obtains pre- multiple
Water jujube flower is sliced；
The rehydration agent is that dimethylbenzene and absolute ethyl alcohol are 0.95~1.05 by volume:0.95~1.05 is mixed to get
Mixed liquor；
12) by pre- rehydration jujube flower that the step 11) obtains slice use successively absolute ethyl alcohol, volumetric concentration 95%,
85%, 75%, 65%, 50% and 30% ethanol water impregnates rehydration, then is transferred to 170~190s of immersion in distilled water, obtains
It is sliced to rehydration jujube flower；
The ethanol water immersion of the absolute ethyl alcohol, volumetric concentration 95%, 85%, 75%, 65%, 50% and 30%
Time stand alone as 170~190s；
13) the siderotil solution immersion 9.5 that the rehydration jujube flower slice mass concentration for obtaining the step 12) is 4%~
First washing after 10.5min, then washed with after 23~27min of hematoxylin dye liquor soaking and dyeing second, then it is molten with saturation picric acid
Third is washed after liquid impregnates 15~30min, obtains dyeing jujube flower slice；
14) the dyeing jujube flower slice that the step 13) obtains is soaked with dimethylbenzene, sucks dimethylbenzene, to the jujube soaked
Flower slice instills neutral gum, and cover plate toasts 28~35min at 37~39 DEG C, obtains jujube flower paraffin section.
Preferably, the mode of dehydration is in the step 2)：First use the ethanol water that volumetric concentration is 30% to described
After fixed jujube flower rinses 1~2 time, then use successively volumetric concentration for 50%, 60%, 70%, 80%, 90% ethanol water
And soaked in absolute ethyl alcohol, it replaces absolute ethyl alcohol and impregnates 25~35min again；
The volumetric concentration be 50%, 60%, 70%, 80%, 90% ethanol water and soaked in absolute ethyl alcohol when
Between stand alone as 0.9~1.1h.
Preferably, the time impregnated in the step 3) is 1.8~2.2h.
Preferably, the number impregnated in the step 4) is 2 times, and the time impregnated every time is 1.4~1.6h/ times.
Preferably, the method for immersion is in the step 5)：Second transparent jujube flower is often impregnated under the conditions of 57~59 DEG C
57~59 DEG C of atoleine is replaced after 2.8~3.2h；The number of the replacement is 2 times.
Preferably, the step 5) or 6) in atoleine be for the first time using paraffin and the mixed liquor that paraffin is used for multiple times；
Described is for the first time 0.95~1.05 with the volume ratio that paraffin is used for multiple times using paraffin:0.95~1.05.
Preferably, the time for opening up piece in the step 9) on glass slide is 30~50min.
Preferably, the temperature for opening up piece in the step 9) on glass slide is 45~55 DEG C.
Preferably, the first time rinsed was 8~12min in the step 13)；
Second time rinsed was 8~12min；
The time that third is rinsed is 5~10min.
Preferably, the usage amount of the neutral gum in the step 14) is 10~20 μ L/ pieces.
The present invention provides a kind of preparation methods of jujube flower paraffin section, include the following steps：1) jujube flower bud is fixed；
2) it is dehydrated；3) the first transparent processing 4) the second transparent processing；5) waxdip；6) it embeds；7) wax is repaiied；8) it is sliced；9) piece is opened up；10) it takes off
Wax；11) rehydration；12) secondary rehydration；13) it dyes；14) cover plate, mounting.It is prepared by jujube flower paraffin section provided by the invention
Method retains the stem section of one section of 0.3~0.5cm in bud base portion, facilitates and clamp tune when stem section is pipetted and embedded in subsequent operation
Whole position, while stem section is able to maintain that bud is horizontal positioned so that bud is parallel to embedded box bottom surface always when embedding, makes to cut
Blastular complete longitudinal section can be switched to when piece, and then facilitates the development change of cell and institutional framework inside observation blastular；By
It is younger tender in jujube tree bud material, select the ethanol water of volume a concentration of 50% to prepare fixer, using low concentration
Ethanol water carry out preparation fixer, shrink material can be prevented, and then the intact form of cell can be kept；Selection is cut
Piece thickness is 8 μm more can completely retain Embryo Sac Development structure in every a piece of wax band.
Meanwhile the present invention using mass concentration be 4% siderotil solution as mordant, enable to material more preferably more fully
Dyeing shortens dyeing time, increases coloring；Color separation is carried out to be saturated picric acid, can be adjusted according to coloring early period
The color separation time obtains institutional framework and clearly, is completely sliced.
Further, dewatering type is after the ethanol water for being first 30% with volumetric concentration rinses 1~2 time, to adopt successively
Ethanol water and soaked in absolute ethyl alcohol with volumetric concentration for 50%, 60%, 70%, 80%, 90%, replace absolute ethyl alcohol again
25~35min is impregnated, selects the ethyl alcohol of the smaller each volumetric concentration of gradient difference to carry out immersion dehydration successively, prevents gradient concentration mistake
Materials microstructure is caused to deform greatly.
Further, during method waxdip provided by the invention all paraffin used be all by volume ratio be 0.95~
1.05:0.95~1.05 new wax and old wax merge, and avoid new wax hardness too big, penetrate into material after solidifying and destroy material
There is broken and fracture with wax band when preventing from being sliced in tissue original structure.Paraffin mixed liquor is selected to be dried at 39~41 DEG C when waxdip
Roasting 35~37h so that waxdip process continues fully, paraffin can be allowed completely into each position of material structure, for post-production gone out
The wax band of high quality lays the foundation.Paraffin section quality height that the present invention is prepared, institutional framework complete display, effect are bright
It shows, operability is strong, repeatability is high, highly practical.Method provided by the invention can spend the jujube flower of different development stage
Flower bud carries out paraffin section, and then observes the variation of blastaea institutional framework and the basic rule of reproductive process in entire jujube flower growth course
Rule.
Description of the drawings
Fig. 1 is in ovule raw period jujube flower paraffin section microphoto for what is be prepared in embodiment 1；
Fig. 2 is in ovule raw period jujube flower paraffin section microphoto for what is be prepared in embodiment 2；
Fig. 3 is in ovule raw period jujube flower paraffin section microphoto for what is be prepared in embodiment 3；
Fig. 4 is in ovule raw period jujube flower paraffin section microphoto for what is be prepared in reference examples 1；
Fig. 5 is the megasporocyte period jujube flower paraffin section microphoto being prepared in embodiment 1；
Fig. 6 is the megasporocyte period jujube flower paraffin section microphoto being prepared in embodiment 2；
Fig. 7 is the megasporocyte period jujube flower paraffin section microphoto being prepared in embodiment 3；
Fig. 8 is the megasporocyte period jujube flower paraffin section microphoto being prepared in reference examples 1.
Specific implementation mode
The present invention provides a kind of preparation methods of jujube flower paraffin section, include the following steps：
1) jujube flower bud is placed in fixer, fixes 24~36h, obtain fixed jujube flower；
The jujube flower bud is with the stem section that length is 0.3~0.5cm；
The fixer be 50% by formalin, glacial acetic acid and volumetric concentration that volumetric concentration is 38% ethyl alcohol
Aqueous solution is 0.95~1.05 by volume:0.95~1.05:17.5~18.5 configurations obtain；
2) the fixation jujube flower in the step 1) is dehydrated, obtains dehydration jujube flower；
3) the dehydration jujube flower in the step 2) is mixed with the first clarifier to immersion and carries out the first transparent processing, obtains the
One transparent jujube flower；
It according to volume ratio is 0.95~1.05 that first clarifier, which is absolute ethyl alcohol and dimethylbenzene,：0.95~1.05 mixing
The mixed liquor of formation；
4) the first transparent jujube flower in the step 3) with dimethylbenzene is mixed to immersion and carries out the second transparent processing, obtain second
Transparent jujube flower and dimethylbenzene system；
5) it is 54~57 DEG C of atoleines, shape to sequentially add dimethylbenzene and temperature to the second transparent jujube flower of the step 4)
At the dimethylbenzene paraffin mixed liquor containing the second transparent jujube flower 35~37h is toasted at 39~41 DEG C, then with every 1.4~1.6h
The rate of 3 DEG C of heating is warming up to 57~59 DEG C of 2.8~3.2h of heat preservation, forms pre- waxdip jujube flower；With 57~59 DEG C after the heat preservation
Atoleine replace the pre- waxdip jujube flower of dimethylbenzene paraffin mixed liquid dipping, obtain waxdip jujube flower；
6) by the step 5) waxdip jujube flower and 57~59 DEG C of atoleine pour into embedded box and wrap simultaneously
It buries, the position of waxdip jujube flower is adjusted after 2~3s, keeps waxdip jujube flower parallel with embedded box bottom surface, it is cooling, obtain embedding jujube flower wax
Block；
7) it carries out the embedding jujube flower wax stone in the step 6) to repair wax, obtains finishing wax stone；
8) the finishing wax stone in the step 7) is sliced, obtains jujube flower slice wax band；
The thickness of the jujube flower slice wax band is 7.5~8.5 μm；
9) the jujube flower slice wax band that the step 8) obtains is transferred in 23~28 DEG C of water, jujube flower slice wax band is existed
Unfold in water, the jujube flower slice wax band after unfolding is transferred on glass slide, and the jujube flower being supported on glass slide is sliced wax band
Piece is opened up, obtains being supported on the jujube flower slice on glass slide；
10) by the jujube flower slice xylene soak dewaxing 13 of being supported on glass slide of obtaining of the step 9)~
17min replaces dimethylbenzene and impregnates 10~15min of dewaxing again, the jujube flower slice to be dewaxed；
11) 9~11min of rehydration is impregnated in the jujube flower slice rehydration agent for the dewaxing for obtaining the step 10), obtains pre- multiple
Water jujube flower is sliced；
The rehydration agent is that dimethylbenzene and absolute ethyl alcohol are 0.95~1.05 by volume:0.95~1.05 is mixed to get
Mixed liquor；
12) by pre- rehydration jujube flower that the step 11) obtains slice use successively absolute ethyl alcohol, volumetric concentration 95%,
85%, 75%, 65%, 50% and 30% ethanol water impregnates rehydration, then is transferred to 170~190s of immersion in distilled water, obtains
It is sliced to rehydration jujube flower；
The ethanol water immersion of the absolute ethyl alcohol, volumetric concentration 95%, 85%, 75%, 65%, 50% and 30%
Time stand alone as 170~190s；
13) the siderotil solution immersion 9.5 that the rehydration jujube flower slice mass concentration for obtaining the step 12) is 4%~
First washing after 10.5min, then washed with after 23~27min of hematoxylin dye liquor soaking and dyeing second, then it is molten with saturation picric acid
Third is washed after liquid impregnates 15~30min, obtains dyeing jujube flower slice；
14) the dyeing jujube flower slice that the step 13) obtains is soaked with dimethylbenzene, sucks dimethylbenzene, to the jujube soaked
Flower slice instills neutral gum, and cover plate toasts 28~35min at 37~39 DEG C, obtains jujube flower paraffin section.
Jujube flower bud is placed in fixer by the present invention, fixes 24~36h, obtains fixed jujube flower；The jujube flower bud band
It is the stem section of 0.3~0.5cm to have length；The fixer is formalin solution, the glacial acetic acid by volumetric concentration for 38%
The ethanol water for being 50% with volumetric concentration is 0.95~1.05 by volume:0.95~1.05:17.5~18.5 with obtained
It arrives.
In the present invention, the jujube flower bud is with the stem section that length is 0.3~0.5cm, preferably 0.4cm.Jujube flower bud
Stem section with above-mentioned length facilitates and clamps stem section in subsequent operation and pipette and adjusted when embedding position, while stem section can be tieed up
It is horizontal positioned to hold bud so that bud is parallel to embedded box bottom surface always when embedding, enables to switch to blastular when slice complete
Longitudinal section, and then facilitate observation blastular inside cell and institutional framework development change.
In the present invention, the ratio of the jujube flower bud and fixer is not particularly limited, and jujube flower bud can be soaked completely
Not in fixer.
The present invention is not particularly limited the kind of the jujube tree used in the jujube flower bud, is using this field routine jujube tree
It can.The jujube flower bud of a diameter of 1.2~3.1mm of winter jujube is used in the embodiment of the present invention.
In the present invention, the jujube flower bud is placed in the set time in fixer as 24~36h, preferably 30h.In this hair
In bright, the ethanol water that formalin, glacial acetic acid and volumetric concentration that the volumetric concentration is 38% are 50% is by volume
Than being preferably 1:1:18.The fixed liquid energy makes the hardening of tissue, is conducive to the progress of slice, and has matchmaker's leaching effect, favorably
It is coloured in tissue.Since jujube tree bud material is younger tender, the ethanol water of volume a concentration of 50% is selected to be distributed as group
Fixer processed can prevent jujube flower shrink material, and then can keep the intact form of cell；Select slice thickness can for 8 μm
More completely to retain Embryo Sac Development structure in every a piece of wax band.
The present invention is not particularly limited using this field the source of the formalin, glacial acetic acid and ethanol water
Conventional commercial product.
After obtaining fixed jujube flower, the present invention is dehydrated the fixed jujube flower, obtains dehydration jujube flower.
In the present invention, the mode of the dehydration is preferably：First use the ethanol water that volumetric concentration is 30% to described solid
After determining jujube flower flushing, then ethanol water and anhydrous second of the volumetric concentration for 50%, 60%, 70%, 80%, 90% are used successively
Alcohol impregnates, and replaces absolute ethyl alcohol and impregnates 25~35min again.The time of the flushing is preferably 1~3min, more preferably 2min.
The time of ethanol water and soaked in absolute ethyl alcohol that the volumetric concentration is 50%, 60%, 70%, 80%, 90% stands alone as
0.9~1.1h, preferably 1h.The time that the replacement absolute ethyl alcohol impregnates again is preferably 30min.Use volumetric concentration for
50%, 60%, 70%, 80%, 90% ethanol water and absolute ethyl alcohol impregnate successively, and the gradient difference of concentration of alcohol is smaller,
Prevent gradient concentration is excessive from materials microstructure being caused to deform.
In the present invention, when the dehydration, the volume of the dehydrating solution of addition is not particularly limited, can be totally submerged solid
Determine jujube flower.
The present invention is 30%, 50%, 60%, 70%, 80%, 90% ethanol water to the volumetric concentration and anhydrous
The source of ethyl alcohol is not particularly limited, using this field conventional commercial absolute ethyl alcohol product configuration.
After obtaining dehydration jujube flower, the dehydration jujube flower is mixed immersion with clarifier and carries out the first transparent processing by the present invention,
Obtain the first transparent jujube flower；It according to volume ratio is 0.95~1.05 that the clarifier, which is absolute ethyl alcohol and dimethylbenzene,：0.95~
1.05 mixed liquors being mixed to form.
In the present invention, the volume ratio of the absolute ethyl alcohol and dimethylbenzene is preferably 1:1.The time of the immersion is preferably
1.8~2.2h, more preferably 2h.
The ratio when present invention mixes the dehydration jujube flower and clarifier is not particularly limited, and the clarifier of addition can
It is totally submerged fixed jujube flower.
The present invention is not particularly limited the source of the absolute ethyl alcohol and dimethylbenzene, using this field conventional commercial product
.
After obtaining the first transparent jujube flower, the described first transparent jujube flower is mixed immersion with dimethylbenzene and carries out second thoroughly by the present invention
Daylight is managed, and the second transparent jujube flower and dimethylbenzene system are obtained.
In the present invention, the number that when second transparent processing impregnates is preferably 2 times.The time of the immersion is preferably
1.4~1.6h/ times, more preferably 1.5h/ times.Jujube flower is dehydrated through secondary transparent processing, tissue inner cell liquid is substituted for alcohol, into
And enter in jujube flower flower bud organization conducive to paraffin in subsequent process.
The ratio that the present invention mixes the described first transparent jujube flower and dimethylbenzene is not particularly limited, the dimethylbenzene energy of addition
Enough it is totally submerged fixed jujube flower.
The present invention is not particularly limited the source of the dimethylbenzene, using this field conventional commercial product.
After obtaining the second transparent jujube flower, the present invention to the described second transparent jujube flower sequentially add dimethylbenzene and temperature be 54~
57 DEG C of atoleine, the dimethylbenzene paraffin mixed liquor containing the second transparent jujube flower of formation toasts 35 at 39~41 DEG C~
37h, then 57~59 DEG C of 2.8~3.2h of heat preservation are warming up to heat up 3 DEG C of rates of every 1.4~1.6h, form pre- waxdip jujube flower；Institute
It states and replaces dimethylbenzene paraffin mixed liquor with 57~59 DEG C of atoleine after keeping the temperature, impregnate pre- waxdip jujube flower, obtain waxdip jujube flower.
In the present invention, the atoleine that dimethylbenzene and temperature are 54~57 DEG C is sequentially added to the described second transparent jujube flower,
The dimethylbenzene paraffin mixed liquor containing the second transparent jujube flower formed.The temperature of the atoleine is preferably 58 DEG C.The temperature
Degree is the paraffin in half curdled appearance for 54~57 DEG C of atoleine.The paraffin of half curdled appearance is conducive to prevent temperature mistake
Height damages material.The fusing point for the atoleine that the temperature is 54~57 DEG C is preferably 56~58 DEG C.
In the present invention, the atoleine preferably uses and is used for multiple times for the first time the mixed liquor of paraffin.It is described for the first time
The volume ratio for using and being used for multiple times paraffin is preferably 0.95~1.05:0.95~1.05, more preferably 1:1.Using making for the first time
With with mixed liquor that paraffin is used for multiple times, can be too big to avoid new wax hardness, penetrate into material after solidifying and destroy material structure
There is broken and fracture with wax band when preventing from being sliced in original structure.
In the present invention, the volume ratio of the dimethylbenzene and atoleine is preferably 0.95~1.05:0.95~1.05, it is more excellent
It is selected as 1:1.The dimethylbenzene paraffin mixed liquor containing the second transparent jujube flower is formed, the present invention contains the second transparent jujube flower by formation
Dimethylbenzene paraffin mixed liquor 35~37h is toasted at 39~41 DEG C.The temperature of the baking is preferably 40 DEG C, the baking
Time is preferably 36h.In the present invention, the baking is so that waxdip process continues fully, paraffin can be allowed completely into jujube flower
The each position of flower bud organization, the wax band that high-quality is made for the later stage lay the foundation.
After the baking, obtained baking systems are warming up to 57 by the present invention with heat up 3 DEG C of rates of every 1.4~1.6h again
After~59 DEG C, 2.8~3.2h is kept the temperature, the pre- waxdip jujube of dimethylbenzene paraffin mixed liquid dipping is replaced with 57~59 DEG C of atoleines
Flower, obtains waxdip jujube flower.In the present invention, the interlude when heating is preferably 1.5h.The heat preservation when
Between preferably 3h.
In the present invention, the method that the atoleine impregnates pre- waxdip jujube flower is preferably：Pre- waxdip jujube flower is existed
It is often impregnated under the conditions of 57~59 DEG C and replaces 57~59 DEG C of atoleine after 2.8~3.2h.57~59 DEG C of liquid stones of the replacement
The number of wax is preferably 2 times.The composition of the atoleine is the same as the atoleine described in said program.
After obtaining waxdip jujube flower, the waxdip jujube flower and 57~59 DEG C of atoleine are poured into embedded box by the present invention simultaneously
In embedded, after 2~3s adjust waxdip jujube flower position, keep waxdip jujube flower parallel with embedded box bottom surface, cool down, embedded
Jujube flower wax stone.
In the present invention, preferably embedded box is preheated before embedding.The temperature of the preheating is preferably 55~60 DEG C, more
Preferably 58 DEG C.
The waxdip jujube flower and 57~59 DEG C of atoleine are poured into embedded box simultaneously, preferably used after 2~3s pre-
Heat keeps waxdip jujube flower parallel with embedded box bottom surface to the position of 80~90 DEG C of dissecting needle adjustment waxdip jujube flower.In the present invention, institute
It is preferably 20~30 DEG C to state cooling temperature, more preferably 25 DEG C.The time of the cooling is 12~36h, more preferably for 24 hours.
After obtaining embedding jujube flower wax stone, the present invention carries out embedded jujube flower wax stone to repair wax, obtains finishing wax stone.
In the present invention, the wax of repairing is preferably：According to jujube flower bud in wax position and direction in the block, wax stone is accomplished and is cut
Face is the small wax stone of rectangle, and the length of side of wax stone section is 2~3 times of jujube flower bud diameter.Embedding jujube flower wax stone is carried out repairing wax,
The wax band for being conducive to that subsequent slice is made to obtain is straight, and convenient segmentation extends piece successively in order, at the same also avoid cut it is cured
Complete embryo is observed in the appearance of process interrupt band and imperfect band, the complete material preserved on per a piece of wax band, guarantee
Capsule growth course.
After obtaining finishing wax stone, the finishing wax stone is sliced by the present invention, obtains jujube flower slice wax band；The jujube flower
The thickness for being sliced wax band is 7.5~8.5 μm, preferably 8 μm.The slice can completely retain blastular in every a piece of wax band
Develop structure.
The present invention is not particularly limited the equipment repaiied used in wax, slice, using this field conventional equipment.
After obtaining jujube flower slice wax band, jujube flower slice wax band is transferred in 23~28 DEG C of water by the present invention, by jujube
Flower slice wax band is unfolded in water, and the jujube flower slice wax band after unfolding is transferred on glass slide, will be supported on glass slide
Jujube flower is sliced wax band and opens up piece, obtains being supported on the jujube flower slice on glass slide.
In the present invention, the temperature of the water is 23~28 DEG C, preferably 25 DEG C.In the present invention, the jujube flower after unfolding is cut
Piece wax band is transferred to glass slide and goes forward first to smear bonding die agent preferably on glass slide, and 1~2 drop distilled water is being added dropwise.The bonding die
The applying amount of agent is 10~20 μ L/ pieces, more preferably 15 μ L/ pieces.The bonding die agent is preferably the mixed solution of egg white and glycerine.
The volume ratio of the egg white and glycerine is preferably 0.9~1.5, more preferably 1:1.In the present invention, the temperature of the exhibition piece is
45~55 DEG C, preferably 50 DEG C.The time of the exhibition piece is preferably 30~50min, more preferably 40min.It opens up piece and completes rear bearing
Moisture on slide is evaporated.
After obtaining being supported on the jujube flower slice on glass slide, the jujube flower being supported on glass slide is sliced and uses by the present invention
Xylene soak 13~17min of dewaxing replaces dimethylbenzene and impregnates 10~15min of dewaxing again, the jujube flower slice to be dewaxed.This
In invention, the time for impregnating dewaxing is 13~17min, preferably 15min.The time for impregnating dewaxing again be 10~
15min, preferably 13min.What surrounding materials became after dewaxing is transparent.
Dewaxed jujube flower slice after, the present invention by the jujube flower slice of the dewaxing with rehydration agent impregnate rehydration 9~
11min obtains pre- rehydration jujube flower slice.The soaking time is preferably 10min.The rehydration agent is dimethylbenzene and absolute ethyl alcohol
It is 0.95~1.05 by volume:0.95~1.05 mixed liquor being mixed to get, preferably 1:1.
After obtaining pre- rehydration jujube flower slice, the present invention by the pre- rehydration jujube flower slice use successively volumetric concentration for
100%, 95%, 85%, 75%, 65%, 50% and 30% ethanol water impregnates rehydration, then is transferred in distilled water and impregnates
170~190s obtains rehydration jujube flower slice.The time impregnated in the distilled water is preferably 180s.The volumetric concentration is
100%, the time that 95%, 85%, 75%, 65%, 50% and 30% ethanol water impregnates stands alone as 170~190s, more
Preferably 180s.
Obtain rehydration jujube flower slice after, the present invention by the rehydration jujube flower slice mass concentration be 4% siderotil solution
9.5~10.5min is impregnated, first rinses, then is rinsed with 23~27min of hematoxylin dye liquor soaking and dyeing, second, then bitter with saturation
Sour solution impregnates 15~30min, and third is rinsed, and obtains dyeing jujube flower slice.
In the present invention, the time that the siderotil solution for being 4% with mass concentration impregnates is 9.5~10.5min, preferably
10min.Described first time rinsed was preferably 8~12min, more preferably 10min.Use mass concentration for 4% siderotil
Solution enables to the more preferably more fully dyeing of jujube flower bud, can shorten dyeing time, increase coloring as mordant.
It is 23~27min, preferably 25min with the time of hematoxylin dye liquor soaking and dyeing in the present invention.Institute's hematoxylin dye
The mass concentration of liquid is 0.4~0.8%, more preferably 0.5%.Nuclear targeting is conducive to as coloring agent using hematoxylin.Institute
The time for stating the second flushing is preferably 8~12min, more preferably 10min.
In the present invention, with saturation picric acid solution impregnate time be 15~30min, preferably 20~25min, more preferably
For 23min.Color separation is carried out using saturation picric acid, it can be according to keeping the institutional framework of acquisition clear, completely.The third is rinsed
Time be preferably 5~10min, more preferably 8min.
Siderotil solution, hematoxylin and the picric source of saturation that the present invention is 4% to the mass concentration be not special
It limits, using this field conventional commercial product.
After obtaining dyeing jujube flower slice, the present invention soaks the dyeing jujube flower slice with dimethylbenzene, sucks dimethylbenzene, to
The jujube flower slice soaked instills neutral gum, and cover plate toasts 28~35min at 37~39 DEG C, obtains jujube flower paraffin section.
In the present invention, after the dyeing jujube flower slice is soaked with dimethylbenzene, preferably by the glass slide back side and dyeing jujube flower
The extra dimethylbenzene of surrounding is sucked with lens wiping paper.Glass slide is preferably shaken after instilling neutral gum, makes neutral gum and diformazan
Benzene is sufficiently mixed.The neutral gum is preferably Canadian neutral gum.The usage amount of the neutral gum is preferably 10~20 μ
L/ pieces, more preferably 15 μ L/ pieces.
In the present invention, the mode of the cover plate is that coverslip side is placed on drop to have on the slice of natural gum, is slowly put down
Coverslip, and air is excluded.
The temperature of heretofore described baking is 37~39 DEG C, more preferably 38 DEG C, time of the baking is 28~
35min, more preferably 30min.
Below in conjunction with the embodiment in the present invention, the technical solution in the present invention is clearly and completely described.It is aobvious
So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
Embodiment 1
From the jujube flower bud of two different flowering time of clip on jujube tree branch, respectively a diameter of 1.21mm, color is
The jujube flower bud and a diameter of 2.00mm of green-yellow, color is the jujube flower bud of yellow green, and is labeled as 1 and No. 2 successively, by this
Two jujube flower buds are respectively according to operations described below step：
The stem section that jujube flower bud base portion is retained to 0.3cm, being placed in fixer, (fixer is：It is 38% by volumetric concentration
The ethanol water that formalin, glacial acetic acid and volumetric concentration are 50% is 0.95 by volume:1.05:17.5 configurations) in
Fixed 36h obtains fixed jujube flower.The ethanol water that volumetric concentration is 30% is first used to rinse 3min obtained fixation jujube flower,
The ethanol water and absolute ethyl alcohol for being successively again 50%, 60%, 70%, 80%, 90% with volumetric concentration respectively impregnate 0.9h, more
It changes absolute ethyl alcohol and impregnates 35min again, obtain dehydration jujube flower.By obtained dehydration jujube flower and clarifier, (clarifier is：Absolute ethyl alcohol
0.95 is pressed with dimethylbenzene:1.05 volume ratio is prepared) mixing immersion 1.8h, obtain the first transparent jujube flower.Thoroughly by obtain first
Bright jujube flower is mixed with dimethylbenzene impregnates 1.4h, discards dimethylbenzene, pours into xylene soak 1.6h from newly, obtain the second transparent jujube flower
With the mixture of dimethylbenzene.Obtain second transparent jujube flower is sequentially added into dimethylbenzene and temperature as 54~57 DEG C of liquid stone
Wax forms the dimethylbenzene paraffin mixed liquor containing the second transparent jujube flower, and 3 DEG C are increased again with every 1.4h after 37h is toasted at 39 DEG C
Rate be warming up to 57 DEG C after, keep the temperature 2.8h, dimethylbenzene paraffin mixed liquor poured out, it is 57 DEG C of atoleine that temperature, which is added,
The atoleine that temperature is 57 DEG C is replaced after often impregnating 3.2h at 57 DEG C, replaces 2 times, obtains waxdip jujube flower.
Obtained waxdip jujube flower and 57 DEG C of atoleine are poured into together and are preheating in 60 DEG C of embedded box, after 2~3s
Jujube flower bud sinks to embedded box bottom, with the dissecting needle adjustment jujube flower bud position of preheating, jujube flower bud is made to be parallel to embedded box
Bottom, the cooling 36h at 30 DEG C, obtains embedding jujube flower wax stone.By obtained embedding jujube flower wax stone according to jujube flower bud in wax stone
In position and direction, wax stone is modified, it is 2~3 times of jujube flower bud diameter to make the length of side of wax stone section, and section is smooth flat
It is whole.It is sliced after repairing wax, slice thickness is 7.5 μm, obtains jujube flower slice wax band.Obtained jujube flower is sliced to the band of wax band
It is face-up laid in clean place in order.Wax band is put into the water that temperature is 23 DEG C with pincet again, wax band is laid in water
On face.10 μ L bonding dies agent are coated on glass slide, and (bonding die agent is：Egg white and glycerine press 0.9:1 volume ratio is prepared), then it is added dropwise 1
Drip distilled water.The slice wax band being laid on the water surface is transferred on the glass slide of oiling bonding die agent, piece is opened up at 45 DEG C
50min obtains being supported on the jujube flower slice on glass slide.The jujube flower slice xylene soak that will be supported on glass slide
13min replaces dimethylbenzene and impregnates 15min again, the jujube flower slice to be dewaxed.The dewaxing jujube flower slice rehydration agent that will be obtained
(rehydration agent is：Dimethylbenzene and absolute ethyl alcohol press 0.95:1 volume ratio is prepared) 9min is impregnated, obtain pre- rehydration jujube flower slice.It will
Obtained pre- rehydration jujube flower slice uses absolute ethyl alcohol and volumetric concentration for 95%, 85%, 75%, 65%, 50% and successively
30% ethanol water respectively impregnates rehydration 190s, then is transferred in distilled water and impregnates 170s, obtains rehydration jujube flower slice.
The siderotil solution that obtained rehydration jujube flower slice mass concentration is 4% is impregnated into 9.5min, after rinsing 12min,
It uses the hematoxylin dye liquor that mass concentration is 0.8% to dye 23min again, rinses 8min, then impregnated with saturation picric acid solution
30min rinses 10min, obtains dyeing jujube flower slice.Obtained dyeing jujube flower slice is soaked with dimethylbenzene, is inhaled with lens wiping paper
The dimethylbenzene for going to jujube flower slice surrounding and glass slide bottom surface is sliced to the jujube flower for soaking dimethylbenzene and 10 μ L Canada neutrality is added dropwise
Natural gum, gently shaking glass slide makes dimethylbenzene and natural gum be sufficiently mixed, and coverslip side, which is placed on drop, to be had on the slice of natural gum,
It slowly puts down coverslip and excludes air, 35min is toasted in 37 DEG C of baking oven, obtain jujube flower paraffin section.Paraffin is cut
Piece is observed under 400 power microscopes, No. 1 bud of concrete outcome as shown in Figure 1, sections observation to bud ovule fall give birth to, can
Ovule is now in judgement to fall raw period；No. 2 buds are as shown in figure 5, sections observation is big to nucleus, the dense big spore of cytoplasm
Primary and secondary cell, it is possible to determine that be in megasporocyte period.The jujube flower that the present invention is prepared it can be seen from Fig. 1 and Fig. 5
Paraffin section institutional framework is clear, complete, is easy to observe, and is able to observe that cell and institutional framework inside entire blastular.
Embodiment 2
From the jujube flower bud of two different flowering time of clip on jujube tree branch, respectively a diameter of 1.50mm, color is
The jujube flower bud and a diameter of 2.23mm of green-yellow, color is the jujube flower bud of yellow green, and is labeled as 3 and No. 4 successively, by this
Two jujube flower buds are respectively according to operations described below step：
The stem section that jujube flower bud base portion is retained to 0.5cm, being placed in fixer, (fixer is：It is 38% by volumetric concentration
The ethanol water that formalin, glacial acetic acid and volumetric concentration are 50% is 1.05 by volume:0.95:18.5 configurations) in
It fixes for 24 hours, obtains fixed jujube flower.The ethanol water that volumetric concentration is 30% is first used to rinse 1min obtained fixation jujube flower,
The ethanol water and absolute ethyl alcohol for being successively again 50%, 60%, 70%, 80%, 90% with volumetric concentration respectively impregnate 1.1h, more
It changes absolute ethyl alcohol and impregnates 25min again, obtain dehydration jujube flower.By obtained dehydration jujube flower and clarifier, (clarifier is：Absolute ethyl alcohol
1.05 are pressed with dimethylbenzene:0.95 volume ratio is prepared) mixing immersion 2.2h, obtain the first transparent jujube flower.Thoroughly by obtain first
Bright jujube flower is mixed with dimethylbenzene impregnates 1.6h, discards dimethylbenzene, pours into xylene soak 1.5h from newly, obtain the second transparent jujube flower
With the mixture of dimethylbenzene.Obtain second transparent jujube flower is sequentially added into dimethylbenzene and temperature as 54~57 DEG C of liquid stone
Wax forms the dimethylbenzene paraffin mixed liquor containing the second transparent jujube flower, and 3 DEG C are increased again with every 1.6h after 35h is toasted at 41 DEG C
Rate be warming up to 59 DEG C after, keep the temperature 3.2h, dimethylbenzene paraffin mixed liquor poured out, it is 59 DEG C of atoleine that temperature, which is added,
The atoleine that temperature is 59 DEG C is replaced after often impregnating 2.8h at 59 DEG C, replaces 2 times, obtains waxdip jujube flower.
Obtained waxdip jujube flower and 59 DEG C of atoleine are poured into together and are preheating in 55 DEG C of embedded box, after 2~3s
Jujube flower bud sinks to embedded box bottom, with the dissecting needle adjustment jujube flower bud position of preheating, jujube flower bud is made to be parallel to embedded box
Bottom, the cooling 12h at 20 DEG C, obtains embedding jujube flower wax stone.By obtained embedding jujube flower wax stone according to jujube flower bud in wax stone
In position and direction, wax stone is modified, it is 2~3 times of jujube flower bud diameter to make the length of side of wax stone section, and section is smooth flat
It is whole.It is sliced after repairing wax, slice thickness is 8.5 μm, obtains jujube flower slice wax band.Obtained jujube flower is sliced to the band of wax band
It is face-up laid in clean place in order.Wax band is put into the water that temperature is 28 DEG C with pincet again, wax band is laid in water
On face.20 μ L bonding dies agent are coated on glass slide, and (bonding die agent is：Egg white and glycerine press 1.5:1 volume ratio is prepared), then it is added dropwise 1
Drip distilled water.The slice wax band being laid on the water surface is transferred on the glass slide of oiling bonding die agent, piece is opened up at 55 DEG C
30min obtains being supported on the jujube flower slice on glass slide.The jujube flower slice xylene soak that will be supported on glass slide
17min replaces dimethylbenzene and impregnates 10min again, the jujube flower slice to be dewaxed.The dewaxing jujube flower slice rehydration agent that will be obtained
(rehydration agent is：Dimethylbenzene and absolute ethyl alcohol press 1.05:1 volume ratio is prepared) 11min is impregnated, obtain pre- rehydration jujube flower slice.
Use absolute ethyl alcohol and volumetric concentration for 95%, 85%, 75%, 65%, 50% and successively obtained pre- rehydration jujube flower slice
30% ethanol water respectively impregnates rehydration 170s, then is transferred in distilled water and impregnates 190s, obtains rehydration jujube flower slice.
The siderotil solution that obtained rehydration jujube flower slice mass concentration is 4% is impregnated into 10.5min, after rinsing 8min,
It uses the hematoxylin dye liquor that mass concentration is 0.4% to dye 27min again, rinses 12min, then impregnated with saturation picric acid solution
15min rinses 5min, obtains dyeing jujube flower slice.Obtained dyeing jujube flower slice is soaked with dimethylbenzene, is sucked with lens wiping paper
The dimethylbenzene of jujube flower slice surrounding and glass slide bottom surface is sliced to the jujube flower for soaking dimethylbenzene and 20 μ L Canada neutral trees is added dropwise
Glue, gently shaking glass slide makes dimethylbenzene and natural gum be sufficiently mixed, and coverslip side, which is placed on drop, to be had on the slice of natural gum, slowly
Coverslip and air is excluded under slow play, toasts 28min in 39 DEG C of baking oven, obtain jujube flower paraffin section.By paraffin section
It is observed under 400 power microscopes, No. 3 buds of concrete outcome, can be with as shown in Fig. 2, sections observation falls to give birth to bud ovule
Judgement is now in ovule and falls raw period；No. 4 buds are as shown in fig. 6, sections observation is big to nucleus, the dense megaspore of cytoplasm
Mother cell, it is possible to determine that be in megasporocyte period.The jujube flower stone that the present invention is prepared it can be seen from Fig. 2 and Fig. 6
Wax biopsy tissues are clear in structure, complete, are easy to observe, and are able to observe that cell and institutional framework inside entire blastular.
Embodiment 3
From the jujube flower bud of two different flowering time of clip on jujube tree branch, respectively a diameter of 1.40mm, color is
The jujube flower bud and a diameter of 2.10mm of green-yellow, color is the jujube flower bud of yellow green, and is labeled as 5 and No. 6 successively, by this
Two jujube flower buds are respectively according to operations described below step：
The stem section that jujube flower bud base portion is retained to 0.4cm, being placed in fixer, (fixer is：It is 38% by volumetric concentration
The ethanol water that formalin, glacial acetic acid and volumetric concentration are 50% is 1 by volume:1:18 configuration) in fix 30h,
Obtain fixed jujube flower.It first uses the ethanol water that volumetric concentration is 30% to rinse 2min obtained fixation jujube flower, then uses successively
The ethanol water and absolute ethyl alcohol that volumetric concentration is 50%, 60%, 70%, 80%, 90% respectively impregnate 1h, replace absolute ethyl alcohol
Solution impregnates 30min again, obtains dehydration jujube flower.By obtained dehydration jujube flower and clarifier, (clarifier is：Absolute ethyl alcohol and diformazan
Benzene presses 1:1 volume ratio is prepared) mixing immersion 2h, obtain the first transparent jujube flower.By the obtain first transparent jujube flower and dimethylbenzene
2h is impregnated in mixing, obtains the mixture of the second transparent jujube flower and dimethylbenzene.Obtain second transparent jujube flower is sequentially added into diformazan
The atoleine that benzene and temperature are 54~57 DEG C, forms the dimethylbenzene paraffin mixed liquor containing the second transparent jujube flower, at 40 DEG C
It is increased after 3 DEG C of rate is warming up to 58 DEG C with 1.5h again after baking 36h, keeps the temperature 3h, dimethylbenzene paraffin mixed liquor is poured out, be added
The atoleine that temperature is 58 DEG C replaces the atoleine that temperature is 58 DEG C after often impregnating 3h at 58 DEG C, replaces 2 times, obtain
Waxdip jujube flower.
Obtained waxdip jujube flower and 58 DEG C of atoleine are poured into together and are preheating in 58 DEG C of embedded box, after 2~3s
Jujube flower bud sinks to embedded box bottom, with the dissecting needle adjustment jujube flower bud position of preheating, jujube flower bud is made to be parallel to embedded box
Bottom cools down for 24 hours at 25 DEG C, obtains embedding jujube flower wax stone.By obtained embedding jujube flower wax stone according to jujube flower bud in wax stone
In position and direction, wax stone is modified, it is 2~3 times of jujube flower bud diameter to make the length of side of wax stone section, and section is smooth flat
It is whole.It is sliced after repairing wax, slice thickness is 8 μm, obtains jujube flower slice wax band.Obtained jujube flower is being sliced the band of wax band just
It is laid in clean place in order up.Wax band is put into the water that temperature is 25 DEG C with pincet again, wax band is laid in the water surface
On.15 μ L bonding dies agent are coated on glass slide, and (bonding die agent is：Egg white and glycerine press 1：1 volume ratio is prepared), then the steaming of 2 drops is added dropwise
Distilled water.The slice wax band being laid on the water surface is transferred on the glass slide of oiling bonding die agent, piece 40min is opened up at 50 DEG C, is obtained
To the jujube flower slice being supported on glass slide.The jujube flower slice xylene soak 15min that will be supported on glass slide replaces two
Toluene impregnates 13min again, the jujube flower slice to be dewaxed.By obtained dewaxing jujube flower slice, with rehydration agent, (rehydration agent is：Two
Toluene and absolute ethyl alcohol press 1:1 volume ratio is prepared) 10min is impregnated, obtain pre- rehydration jujube flower slice.The pre- rehydration jujube that will be obtained
Flower slice use successively absolute ethyl alcohol and volumetric concentration for 95%, 85%, 75%, 65%, 50% and 30% ethanol water
It is each to impregnate rehydration 180s, then be transferred in distilled water and impregnate 180s, obtain rehydration jujube flower slice.
The siderotil solution that obtained rehydration jujube flower slice mass concentration is 4% is impregnated into 10min, after rinsing 10min, then
The hematoxylin dye liquor for being 0.5% with mass concentration dyes 25min, rinses 10min, then impregnate 23min with saturation picric acid solution,
8min is rinsed, dyeing jujube flower slice is obtained.Obtained dyeing jujube flower slice is soaked with dimethylbenzene, sucking jujube flower with lens wiping paper cuts
Around piece and the dimethylbenzene of glass slide bottom surface, it is sliced to the jujube flower for soaking dimethylbenzene and 15 μ L Canada neutral gums is added dropwise, gently
The light glass slide that shakes makes dimethylbenzene and natural gum be sufficiently mixed, and coverslip side, which is placed on drop, to be had on the slice of natural gum, is slowly put
Lower coverslip simultaneously excludes air, toasts 30min in 38 DEG C of baking oven, obtains jujube flower paraffin section.By paraffin section 400
It is observed under power microscope, No. 5 buds of concrete outcome are as shown in figure 3, sections observation falls to give birth to bud ovule, it is possible to determine that this
When fallen raw period in ovule；For No. 6 buds as shown in fig. 7, sections observation is big to nucleus, the dense macrospore of cytoplasm is thin
Born of the same parents, it is possible to determine that be in megasporocyte period.The jujube flower paraffin that the present invention is prepared it can be seen from Fig. 3 and Fig. 7 is cut
Piece institutional framework is clear, complete, is easy to observe, and is able to observe that cell and institutional framework inside entire blastular.
Reference examples 1
From the jujube flower bud of two different flowering time of clip on jujube tree branch, respectively a diameter of 1.42mm, color is
The jujube flower bud and a diameter of 2.12mm of green-yellow, color is the jujube flower bud of yellow green, and is labeled as 7 and No. 8 successively, is used
The production method of paraffin section in the prior art makes jujube flower paraffin section, and concrete operation method is as follows：
1 fixes：Jujube flower bud is placed in fixer, and (fixer is the FAA of improvement：Fixer is：Volumetric concentration is 38%
Formaldehyde：Glacial acetic acid：Volumetric concentration is 50% ethyl alcohol=1：1：18) 30h is fixed in, is placed on filter paper after blotting fixer, will be spent
Flower bud, which is respectively placed in the 10mL centrifuge tubes of corresponding grade label, carries out next stage operation.
2 dehydrations：According to -90% wine of -80% alcohol (1h) of 50% alcohol (1h) -70% alcohol (1h) of -60% alcohol (1h)
The sequence of -100% alcohol (1h) of smart (1h) -95% alcohol (1h) -100% alcohol (1h) and time are dehydrated.
3 is transparent:According to the sequence of+1/2 absolute ethyl alcohol of 1/2 dimethylbenzene (1.5h)-pure dimethylbenzene (2h)-pure dimethylbenzene (2h)
Carrying out transparent processing with the time, (it is 1 that+1/2 absolute ethyl alcohol of 1/2 dimethylbenzene, which is volume ratio,:1 dimethylbenzene and absolute ethyl alcohol
Mixed liquor).
4 waxdips：Material after transparent be placed in 1/2 dimethylbenzene+1/2 it is pure it is cured in, placed in 38 DEG C~40 DEG C constant temperature ovens
After 36h, be gradually warming up to after 60 DEG C according to each gradient for increasing by 3 DEG C and keep 2h, then by material it is hyperphoric twice in it is pure it is cured in,
Each 1.5h.
5 embeddings：Thermostat water bath temperature setting is 55 DEG C, and one piece of smooth glass plate in surface is selected to be placed in water-bath
On, small square box made of ready brown paper folds will be shifted to an earlier date and be placed on glass plate, advance demand flag is good straight with difference on capsule
The corresponding number of material of diameter size, to facilitate subsequent statistics；Prepare one piece of Baking out on alcolhol burner simultaneously
Dissecting needle；When embedding, the small beaker of waxdip is rapidly taken out from constant temperature oven, and paraffin and material are poured into small square box together
In, material is uniformly arranged in carton by 2~3s after material sinks to cassette bottom, with dissecting needle, after paraffin, is positioned over
Ventilation can carry out repairing wax and slice after 1~2 day.
6 repair wax：After the paraffin mass of box and embedded material that brown paper is made is separated, according to the size and reality of material
Paraffin mass is divided the fritter of squarely by the requirement of slice.
7 slices：It is rapidly performed by slice, slice thick after the rectangular fritter paraffin for being embedded with material is fixed on slicer
Degree is 8 μm.
8 bonding dies and drying：The glass slide for being coated with bonding die agent in advance is ready to, upper 2~3 is dripped in the above and drips, it will
Water droplet is uniformly distributed on glass slide, and the material cut is divided into suitable length according to the length of glass slide, is being carried successively
It is flattened on slide, is placed at 55 DEG C and dries 30min.
9 dyeing：Material after dimethylbenzene dewaxes, be sequentially placed into absolute ethyl alcohol and volumetric concentration be 95%, 85%, 75%,
65%, 50% and 30% ethanol water impregnates rehydration, and the time impregnated in each gradient concentration alcohol is 3~5min, then
It is transferred to 3~5min of immersion in distilled water.Obtain rehydration jujube flower slice；It is 4% iron that obtained rehydration slice, which is placed in volumetric concentration,
10min is impregnated in alum solution, after with flowing water rinse 10min；It is placed in again in 0.5% hematoxylin dye liquor and impregnates 10min, after afterflow
Water rinses 5min, is finally placed in 4% 10~20min of siderotil solution color separation (microscopy determines color separation degree in soaking process), terminates
Afterwards indigo plant is returned with tap water 10~15min of flushing to material.
10 microscopies：After dyeing, glass slide is bedewed in dimethylbenzene, glass slide bottom and surrounding are wiped with lens wiping paper
Extra dimethylbenzene instills one in glass slide center and neutral gum of putting on airs is added dropwise, and gently shaking slide makes natural gum and dimethylbenzene fill
Divide mixing, please put coverslip from glass slide one end, be subsequently placed in 38 DEG C of baking oven and dry, obtain winter jujube flower tissue forever
Slice long, can be observed under the microscope at any time.
Paraffin section is observed under 400 power microscopes, concrete outcome can be seen as shown in Fig. 4,8 by Fig. 4 and 8
Go out, the jujube flower paraffin section picture blur obtained using prior art preparation is not easy to observe.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of preparation method of jujube flower paraffin section, includes the following steps：
1) jujube flower bud is placed in fixer, fixes 24~36h, obtain fixed jujube flower；
The jujube flower bud is with the stem section that length is 0.3~0.5cm；
The fixer be 50% by formalin, glacial acetic acid and volumetric concentration that volumetric concentration is 38% ethyl alcohol it is water-soluble
Liquid is 0.95~1.05 by volume:0.95~1.05:17.5~18.5 preparations obtain；
2) the fixation jujube flower in the step 1) is dehydrated, obtains dehydration jujube flower；
3) the dehydration jujube flower in the step 2) with clarifier is mixed to immersion and carries out the first transparent processing, obtain the first transparent jujube
Flower；
It according to volume ratio is 0.95~1.05 that first clarifier, which is absolute ethyl alcohol and dimethylbenzene,：0.95~1.05 is mixed to form
Mixed liquor；
4) the first transparent jujube flower in the step 3) with dimethylbenzene is mixed to immersion and carries out the second transparent processing, it is transparent to obtain second
Jujube flower and dimethylbenzene system；
5) it is 54~57 DEG C of atoleines to sequentially add dimethylbenzene and temperature to the second transparent jujube flower of the step 4), formation
Dimethylbenzene paraffin mixed liquor containing the second transparent jujube flower toasts 35~37h at 39~41 DEG C, then is heated up with every 1.4~1.6h
3 DEG C of rate is warming up to 57~59 DEG C of 2.8~3.2h of heat preservation, forms pre- waxdip jujube flower；With 57~59 DEG C of liquid after the heat preservation
Body paraffin replaces dimethylbenzene paraffin mixed liquor, impregnates pre- waxdip jujube flower, obtains waxdip jujube flower；
6) by the step 5) waxdip jujube flower and 57~59 DEG C of atoleine pour into embedded box and embed, 2~3s
The position for adjusting waxdip jujube flower afterwards, keeps waxdip jujube flower parallel with embedded box bottom surface, cooling, obtains embedding jujube flower wax stone；
7) it carries out the embedding jujube flower wax stone in the step 6) to repair wax, obtains finishing wax stone；
8) the finishing wax stone in the step 7) is sliced, obtains jujube flower slice wax band；
The thickness of the jujube flower slice wax band is 7.5~8.5 μm；
9) the jujube flower slice wax band that the step 8) obtains is transferred in 23~28 DEG C of water, in water by jujube flower slice wax band
To unfold, the jujube flower slice wax band after unfolding is transferred on glass slide, and the jujube flower being supported on glass slide slice wax band is opened up piece,
Obtain being supported on the jujube flower slice on glass slide；
10) the jujube flower slice xylene soak 13~17min of dewaxing being supported on glass slide for obtaining the step 9), more
It changes dimethylbenzene and impregnates 10~15min of dewaxing again, the jujube flower slice to be dewaxed；
11) 9~11min of rehydration is impregnated in the jujube flower slice rehydration agent for the dewaxing for obtaining the step 10), obtains pre- rehydration jujube
Flower slice；
The rehydration agent is that dimethylbenzene and absolute ethyl alcohol are 0.95~1.05 by volume：0.95~1.05 mixing being mixed to get
Liquid；
12) by pre- rehydration jujube flower that the step 11) obtains slice use successively absolute ethyl alcohol, volumetric concentration 95%, 85%,
75%, 65%, 50% and 30% ethanol water impregnates rehydration, then is transferred to 170~190s of immersion in distilled water, obtains rehydration
Jujube flower is sliced；
The absolute ethyl alcohol, volumetric concentration 95%, 85%, 75%, 65%, 50% and 30% ethanol water impregnate when
Between stand alone as 170~190s；
13) the siderotil solution immersion 9.5 that the rehydration jujube flower slice mass concentration for obtaining the step 12) is 4%~
First washing after 10.5min, then washed with after 23~27min of hematoxylin dye liquor soaking and dyeing second, then it is molten with saturation picric acid
Third is washed after liquid impregnates 15~30min, obtains dyeing jujube flower slice；
14) the dyeing jujube flower slice that the step 13) obtains is soaked with dimethylbenzene, neutral tree is instilled to the jujube flower slice soaked
Glue, cover plate toast 28~35min at 37~39 DEG C, obtain jujube flower paraffin section.
2. the preparation method of jujube flower paraffin section according to claim 1, which is characterized in that be dehydrated in the step 2)
Mode is：After first using the ethanol water that volumetric concentration is 30% to rinse the fixed jujube flower 1~2 time, then body is used successively
A concentration of 50%, 60%, 70%, 80%, 90% ethanol water of product and soaked in absolute ethyl alcohol are replaced absolute ethyl alcohol and are impregnated again
25~35min；
The time of ethanol water and soaked in absolute ethyl alcohol that the volumetric concentration is 50%, 60%, 70%, 80%, 90% is only
It stands as 0.9~1.1h.
3. the preparation method of jujube flower paraffin section according to claim 1, which is characterized in that impregnated in the step 3)
Time is 1.8~2.2h.
4. the preparation method of jujube flower paraffin section according to claim 1, which is characterized in that impregnated in the step 4)
Number is 2 times, and the time impregnated every time is 1.4~1.6h/ times.
5. the preparation method of jujube flower paraffin section according to claim 1, which is characterized in that impregnated in the step 5)
Method is：Second transparent jujube flower is often impregnated under the conditions of 57~59 DEG C and replaces 57~59 DEG C of atoleine after 2.8~3.2h；
The number of the replacement is 2 times.
6. the preparation method of jujube flower paraffin section according to claim 1, which is characterized in that the step 5) or 6) in liquid
Body paraffin is for the first time using paraffin and the mixed liquor that paraffin is used for multiple times；
Described is for the first time 0.95~1.05 with the volume ratio that paraffin is used for multiple times using paraffin：0.95~1.05.
7. the preparation method of jujube flower paraffin section according to claim 1, which is characterized in that glass slide in the step 9)
The time of upper exhibition piece is 30~50min.
8. the preparation method of jujube flower paraffin section according to claim 1, which is characterized in that glass slide in the step 9)
The temperature of upper exhibition piece is 45~55 DEG C.
9. the preparation method of jujube flower paraffin section according to claim 1, which is characterized in that first in the step 13)
The time of flushing is 8~12min；
Second time rinsed was 8~12min；
The time that third is rinsed is 5~10min.
10. according to the preparation method of jujube flower paraffin section as described in any one of claim 1 to 9, which is characterized in that the step
It is rapid 14) in neutral gum usage amount be 10~20 μ L/ pieces.
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| CN113039955A (en) * | 2021-01-22 | 2021-06-29 | 韶关学院 | Method for inhibiting loquat flowering |
| CN113702131A (en) * | 2021-09-06 | 2021-11-26 | 海南大学 | Paraffin section of camellia oleifera flower bud and preparation method and application thereof |
| CN114235536A (en) * | 2021-12-24 | 2022-03-25 | 贵州大学 | Slice preparation method for observing lignin deposition of two-year-old masson pine stems |
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| CN113702131A (en) * | 2021-09-06 | 2021-11-26 | 海南大学 | Paraffin section of camellia oleifera flower bud and preparation method and application thereof |
| CN114235536A (en) * | 2021-12-24 | 2022-03-25 | 贵州大学 | Slice preparation method for observing lignin deposition of two-year-old masson pine stems |
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Inventor after: Li Yingyue Inventor after: Huang Feiyi Inventor after: Cui Yanhong Inventor after: Pang Xiaoming Inventor after: Li Xiang Inventor before: Li Yingyue Inventor before: Huang Feiyi Inventor before: Cui Yanhong Inventor before: Pang Xiaoming Inventor before: Li Xiang |
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Application publication date: 20180727 |