CN105699142B - Paraffin section preparation method for the close hard vegetable material of quality - Google Patents
Paraffin section preparation method for the close hard vegetable material of quality Download PDFInfo
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- CN105699142B CN105699142B CN201610090674.4A CN201610090674A CN105699142B CN 105699142 B CN105699142 B CN 105699142B CN 201610090674 A CN201610090674 A CN 201610090674A CN 105699142 B CN105699142 B CN 105699142B
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- 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
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- 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
- G01N1/36—Embedding or analogous mounting of samples
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Abstract
The present invention relates to plant sample tissue paraffin section de technical field, especially a kind of paraffin section preparation method for vegetable material development later stage seeds such as the close hard rice wheats of quality.A kind of paraffin section preparation method for the close hard vegetable material of quality, comprise the following steps:（1）It is fixed：Take the plant seed material of different times after pollinating to carry out rip cutting or crosscutting, be put into room temperature in fixer and fix 3~5h；（2）Dehydration：Above-mentioned fixer is suctioned out using liquid-transfering gun, the alcohol that gradient is 30%, 50%, 75%, 95% and 100% concentration is successively sequentially added and is dehydrated；（3）It is transparent：After above-mentioned alcohol is suctioned out using imbibition rifle, clarifier is made using chloroform；（4）Waxdip；（5）Embedding and section.The present invention is workable, method is easy, economical and practical, can solve the problem that the frangible problem of development later stage seed section, easily cuts out the complete paraffin section of development later stage seed.
Description
Technical field
It is especially a kind of for the close hard rice of quality the present invention relates to plant sample tissue paraffin section de technical field
The vegetable materials such as wheat, such as the paraffin section preparation method of Wheat Development later stage seed.
Background technology
Paraffin section technology is to be engaged in the basic test technical method of Developmental Biology and Histochemical studies, and micro-
Observation of plant institutional framework common technology.The botany research field technology research phytomorph build up, plant hybridization breeding,
The cultivation and discriminating of rare plant etc. extensive application.In addition, for animal and plant sample carry out histochemical stain,
Carry out immunohistochemical study and carry out the research such as in situ hybridization, all be unable to do without and paraffin section is carried out to animal and plant sample tissue
Make, and paraffin section make excellent Degree of Accord Relation to correlative study result of the test order of accuarcy.Therefore, paraffin section
Technology is that the basic test technology of animal and plant Developmental Biology and Histochemical studies is carried out in life science.
Wheat seed development substantially undergone since chasmogamy kernel grouting initial stage, milk stage, stage of wax ripeness, the stage of yellow ripeness and
Full ripe stage.As kernel grouting process continues, change from small to big in wheat seed form, as starch in seed and albumen gradually accumulate
Tired, wheat seed gradually develops into the close hard solid state endosperm of quality by the liquid endosperm of Early filling stage.For seeds such as rice wheats
The preparation method of paraffin section, by literature search（Kang Haiqi etc., 2010）The method inquired is according to conventional mostly
Plant paraffin microsection manufacture universal method, and reported that the paraffin section method that correlative study uses can only cut out grouting early stage
Seed paraffin section.Due to wheat seed Early filling stage, kernel texture is relatively soft to be easier to cut out complete paraffin section, and small
Wheat Grain Development later stage kernel texture is closely hard, frangible during section more difficult to cut out entire kernel paraffin section.After Wheat Development
The main reason for section of phase seed is difficult has two aspects：On the one hand for seed material, after in wheat seed development
Phase starch and a large amount of accumulation of protein and moisture content of kernels reduce, and cause seed material to be hardened, endosperm tissue quality becomes
It is fine and close.On the other hand in dicing method, traditional paraffin section preparation methods are summarised as dehydration of alcohol, and dimethylbenzene is transparent, then by
The method of level waxdip.Because dimethylbenzene can be contracted thereby to become hard organization material, thus cause originally fine and close seed material waxdip meeting
It is more difficult, and the traditional paraffin section preparation methods waxdip time is too short, and this results in paraffin and is not completely immersed in seed histocyte
Inside, therefore material is extremely fragile when doing seed section, it is difficult to cut out the paraffin section of entire kernel.In rice grain paraffin
It is firm etc. in knowing on microsection manufacture（2010）Attempt to carry out seed material sofening treatment, i.e. the absolute ethyl alcohol of 1/2 glycerine+1/2 mixes
Instead of dimethylbenzene, clearing time is one week, and as a result seed can become transparent, but be not easy to cut into slices, and its method can not be cut out
More than 12d entire kernel paraffin section after pollination.Kang Haiqi etc. softens seed using 0.5%~15% hydrofluoric acid solution, when
Between from 30min to 30 d, but material is still not easy to cut into slices；It is allowed to soften if rice is boiled, endosperm starch will become to be gelatinized shape, this
Kind method can only also be switched to the seed of 11d after pollination.
Therefore, the frangible, researcher of Wheat Development later stage seed section is not easy to cut out the complete of Wheat Development later stage seed
The problem of whole paraffin section is one anxious to be resolved.
The content of the invention
It is an object of the invention to provide it is a kind of it is workable, method is easy, economical and practical, can solve the problem that development later stage
Seed cuts into slices frangible problem, and the complete paraffin section for easily cutting out development later stage seed is used for quality closely hard vegetable material
Paraffin section preparation method.
The invention discloses a kind of paraffin section preparation method for the close hard vegetable material of quality, it is characterised in that
Comprise the following steps:
(1) it is, fixed:
Take the plant seed material of different times after pollinating to carry out rip cutting or crosscutting, be put into the fixer in container, make
Vacuumized with vacuum desiccator, all sink to container bottom to all seed materials, room temperature fixes 3~5h；
This step points for attention：
(1) it is, the chloroform that can corrode plastics due to making the clarifier that uses of section later stage, thus fixes and open from material
Beginning should use vial, and should not use plastic centrifuge tube.
（2）, should be according to subsequent experimental specific requirement come from suitable fixer, if making the paraffin in situ hybridization
Section, needs to use（4% paraformaldehyde+0.1%（v/v）DEPC）As fixer composition.In addition, needed according to experiment from conjunction
Suitable fixer, for example the paraffin section in situ hybridization is made, it need to be fixed using 4% paraformaldehyde, and add 0.1%（v：
v）DEPC；Such as need to dye observation of plant histocyte, and plant tissue need to be preserved for a long time, FAA fixers can be used；As needed
Carry out DAPI dyeing（DAPI is a kind of fluorescent dye, and specifically DNA can be dyed, can be with observation group under fluorescence
Knit interior nucleus）, Kano fixer can be used.
(3), seed must carry out rip cutting or crosscutting, and otherwise fixer is difficult to enter, and can cause excessively fixed outside seed
And it is internal also unlocked, while the steps such as dehydration also after influence is transparent.
（Two）, dehydration：
Above-mentioned fixer is suctioned out using liquid-transfering gun, it is dense for 30%, 50%, 75%, 95% and 100% successively to sequentially add gradient
The alcohol of degree is dehydrated：30% dehydration of alcohol 3~5 times, an alcohol is changed every 20~30min；50% dehydration of alcohol 3~5
It is secondary, change an alcohol every 20~30min；Sample is transferred on shaking table or shaken every 8~12min afterwards and is held
Device, 75% dehydration of alcohol 3~5 times, an alcohol is changed every 40~50min；95% dehydration of alcohol 3~5 times, every 40~
50min changes an alcohol；100% absolute alcohol is dehydrated 3~5 times, and an alcohol is changed every 40~50min；
This step points for attention：
(1), change after 75% alcohol it can be found that the chlorophyll in Early filling stage seed fruit kind skin is gradually taken out by alcoholic extract
Come, close to the alcohol greening of bottom of bottle, therefore rocked using shaking table, to keep alcohol uniform, be smoothed out seed dehydration.
(2), this step can also be without using shaking table, but needs to shake container every 10min or so.To for several times finally
No chlorophyll is extracted out when changing alcohol, shows that seed dehydration is complete.
Conventional method often walks dewatering time and is generally 1~2h, and the step extends dewatering time compared with conventional method, together
When using shaking table make dehydration more smooth, and easily determine whether seed dehydration is completed.
（Three）, it is transparent：
After above-mentioned alcohol is suctioned out using imbibition rifle, clarifier is made using chloroform, addition volume ratio is alcohol：Chloroform=3：1
3~4h of solution left standstill, it is evacuated to all material and sinks to the bottom；Solution is suctioned out using imbibition rifle afterwards, addition volume ratio is alcohol：
Chloroform=1：1 solution, 3~4h is stood, all material is evacuated to and sinks to the bottom；Reuse imbibition rifle to suction out solution, add volume
Than for alcohol：Chloroform=1：3 solution, 3~4h is stood, all material is evacuated to and sinks to the bottom；Imbibition rifle is reused to inhale solution
Go out, add pure chloroform and stand 2~3d, be evacuated to all material and sink to the bottom；
This step points for attention：
（1）, chloroform will not make Material shrinkage be hardened, therefore use chloroform instead and make clarifier.But chloroform seepage velocity is slower, by
Being in the milk in wheat seed, later stage material itself is harder, water content is relatively low, therefore this step clearing time will more than conventional process time
It is long.
（2）, due to chloroform density it is larger, every time improve chloroform ratio when, material can all be floated to liquid level, so every time
Need to vacuumize to promote clarifier to enter inside material structure.
（3）, at the end of transparent step, it can be found that the grouting later stage（35d after such as pollinating）Material can also against light source
Printing opacity, show that the seed step is transparent good.
The step uses chloroform instead and makees clarifier, and significantly extends clearing time, can accomplish not make Material shrinkage be hardened
It is and transparent complete.
（Four）, waxdip：
After using imbibition rifle, the above-mentioned pure chloroform is suctioned out, addition volume ratio is chloroform：Paraffin=3：1 mixed liquor,
4~5h is incubated at 40~42 DEG C；Afterwards, after being suctioned out above-mentioned chloroform and paraffin mixed liquor using imbibition rifle, adding volume ratio is
Chloroform：Paraffin=1：1,4~5h is incubated at 40~42 DEG C；Afterwards, above-mentioned chloroform and paraffin mixed liquor are suctioned out using imbibition rifle
Afterwards, it is chloroform to add volume ratio：Paraffin=1：3 mixed liquor, 4~5h is incubated at 45~47 DEG C；Before 21d after pollination
Material, 3~4d is incubated at 55~57 DEG C using paraffin refined wax；For the later materials of 21d after pollination, using paraffin refined wax 55
~57 DEG C are incubated 3~5d, and a paraffin refined wax is changed during waxdip；
This step points for attention：
（1）, wheat seed starch itself and protein content it is higher, it is especially even more so to the Grain Development later stage, cause to soak
Wax is very difficult.Therefore the waxdip time must be extended, and suitable temperature must be kept during waxdip, temperature is too high to be not only broken up
Structure inside seed, and higher than 60 DEG C when be unfavorable for preserve organization internal bioactive substance, influence SABC
As a result, also tissue can be made to be hardened, become fragile, also results in the overall loose, disintegration of seed, therefore, keep 55 DEG C of waxdips, make at paraffin
In slush state.
（2）, waxdip it is complete after, it can be found that material becomes micro- green or white translucent bowlder-like.
The step keeps relatively low waxdip temperature, but significantly extends the waxdip time, can make in seed histocyte
Complete waxdip, while can ensure that material is complete and does not destroy internal structure, moreover it is possible to preserve the bioactive substance of organization internal.
As shown in Fig. 2 be the inventive method and the wheat seed cross section comparison diagram after conventional method waxdip, A, B in figure
Be expressed as wheat spend rear 21d and 28d seed use this method waxdip after effect, C, D be respectively wheat spend rear 21d and
28d seed uses the effect after conventional method waxdip.It is as shown in Fig. 2 complete using the wheat seed waxdip after this method waxdip
Entirely, be translucent shape, and not having starch during section drops, and wheat seed waxdip is insufficient after using conventional method waxdip, cuts
Starch is had during piece to drop.
（Five）, embedding and section：
Used seed material is embedded according to a conventional method with carton or FFPE box, before 21d after pollination
Material directly can cut into slices on paraffin slicing machine according to a conventional method, must be used after material FFPE later 21d after pollination
Blade cuts out material cross-section, and then by wax stone back-off, 4~6 DEG C of softenings overnight, are then cut into slices again in DEPC water, above-mentioned
All seed materials can be cut into the section of 8~25 μm of thickness range.
This step adds DEPC water bating steps for Grain Development later stage material, makes the Grain Development later stage basic
Ripe seed can also cut out whole slices.
Through paraffin section made of the method after roasting piece, dewaxing and rehydration, it can be entered according to follow-up study experiment purpose
Enter ensuing experimental implementation.
As shown in figure 3, be the contrast picture of the wheat seed paraffin section cut out using the inventive method and conventional method,
Shown in figure：A, B, C be respectively wheat spend rear 21,28, the paraffin section that is cut out using this method of 35d seed, D, E, F difference
Spend rear 21 for wheat, 28, the paraffin section that is cut out using conventional method of 35d seed.
As shown in figure 3, the complete paraffin section of wheat seed development later stage can be cut out using the inventive method, and use
The paraffin section that conventional method is cut out is substantially all imperfect.
The present invention basic functional principle be：
Paraffin section technology is to do one of life science common technology, due in the development of plants later stage such as wheat seed
Starch and protein accumulation, moisture content of kernels is reduced in addition, and seed material can be caused to be hardened, and grain endosperm tissue becomes fine and close.
Using traditional paraffin section preparation methods seed Material shrinkage can be made to be hardened, material waxdip is very difficult, thus easily broken during section, very
Difficulty cuts out complete paraffin section.To solve this problem, the present invention changes clarifier, extended by repeatedly attempting and improving
Clearing time, waxdip time and temperature is adjusted, propose to be directed to quality closely hard vegetable material（Wheat Development later stage seed）Stone
Wax microsection manufacture new method.Wheat seed can be made from Early filling stage to each period during seed complete ripeness using this method
Entire kernel paraffin section, it can be applied in wheat seed Developmental Biology and histochemical correlative study.
By the improvement of constantly bringing forth new ideas to Wheat Development later stage seed paraffin section method, the present invention is for after Wheat Development
The close hard characteristic of phase kernel texture, is proposed by using chloroform as clarifier, and extends clearing time to 3d, and is adjusted
Whole waxdip time and temperature, can make paraffin be completely immersed in seed organization internal, and it is easy to solve the section of Wheat Development later stage seed
Broken problem, researcher can be made to be easy to cut out the complete paraffin section of Wheat Development later stage seed.
As shown in figure 4, the microphoto for wheat seed different times paraffin section after I2-KI is dyed, A, B in figure,
C, D be respectively wheat spend rear 14,21,28, the microphoto taken pictures immediately after I2-KI is dyed of 35d seed paraffin section, E,
F, G, H be respectively wheat spend rear 14,21,28, the microphoto taken pictures after I2-KI dyes 48h of 35d seed paraffin section.
Each period complete paraffin section after wheat seed is pollinated has been produced using the inventive method.If Fig. 4 is to use this method
The section of making is taken pictures immediately after I2-KI is dyed（A~D in figure）After dyeing 48h（E~H in figure）Take pictures to obtain micro-
Photo.From figure it will be clear that in wheat seed albuminous cell starch distribution（A~D in figure）And clearly cell
Structure（E~H in figure）.
Compared with prior art, the present invention is that one kind is workable, method is easy, economical and practical, can solve the problem that development
Later stage seed cuts into slices frangible problem, and the complete paraffin section for easily cutting out development later stage seed is used for quality closely hard plant
The paraffin section preparation method of material.
Brief description of the drawings
Fig. 1 is implementation steps flow chart of the present invention.
It is the inventive method and the wheat seed cross section comparison diagram after conventional method waxdip that Fig. 2, which is, A, B difference table in figure
Being shown as wheat spends rear 21d and 28d seed to use the effect after this method waxdip, and C, D are respectively that wheat spends rear 21d and 28d
Seed uses the effect after conventional method waxdip.
Fig. 3 is the contrast picture of the wheat seed paraffin section cut out using the inventive method and conventional method, institute in figure
Show：A, B, C be respectively wheat spend rear 21,28, the paraffin section that is cut out using this method of 35d seed, D, E, F are respectively wheat
Spend rear 21,28, the paraffin section that is cut out using conventional method of 35d seed.
Fig. 4 is microphoto of the wheat seed different times paraffin section after I2-KI is dyed, and A, B, C, D distinguish in figure
Spend rear 14 for wheat, 21,28, the microphoto taken pictures immediately after I2-KI is dyed of 35d seed paraffin section, E, F, G, H points
Not Wei wheat spend rear 14,21,28, the microphoto taken pictures after I2-KI dyes 48h of 35d seed paraffin section.
Embodiment
Embodiment 1:
After pollinating 7,14,21,28,35d each 5 of fresh wheat seed is taken respectively, vial is put into after crosscutting, is added
The paraformaldehyde fixers of 5ml 4%, vacuumize fixed seed.Next operated according to this method, you can cut out wheat seed development
Each period complete paraffin section.
Reference picture 1- Fig. 4, a kind of paraffin section preparation method for the close hard vegetable material of quality, including following step
Suddenly:
(1) it is, fixed:
After pollinating 7,14,21,28,35d each 5 of fresh wheat seed is taken respectively, vial is put into after crosscutting, is added
The paraformaldehyde fixers of 5ml 4%, are vacuumized using vacuum desiccator, all sink to container bottom to all seed materials, room temperature is solid
Determine 4h；
This step points for attention：
(1) it is, the chloroform that can corrode plastics due to making the clarifier that uses of section later stage, thus fixes and open from material
Beginning should use vial, and should not use plastic centrifuge tube.
（2）, should be according to subsequent experimental specific requirement come from suitable fixer, if making the paraffin in situ hybridization
Section, needs to use（4% paraformaldehyde+0.1%（v/v）DEPC）As fixer composition.In addition, needed according to experiment from conjunction
Suitable fixer, for example the paraffin section in situ hybridization is made, it need to be fixed using 4% paraformaldehyde, and add 0.1%（v：
v）DEPC；Such as need to dye observation of plant histocyte, and plant tissue need to be preserved for a long time, FAA fixers can be used；As needed
Carry out DAPI dyeing（DAPI is a kind of fluorescent dye, and specifically DNA can be dyed, can be with observation group under fluorescence
Knit interior nucleus）, Kano fixer can be used.
(3), seed must carry out rip cutting or crosscutting, and otherwise fixer is difficult to enter, and can cause excessively fixed outside seed
And it is internal also unlocked, while the steps such as dehydration also after influence is transparent.
（Two）, dehydration：
Above-mentioned fixer is suctioned out using liquid-transfering gun, it is dense for 30%, 50%, 75%, 95% and 100% successively to sequentially add gradient
The alcohol of degree is dehydrated：30% dehydration of alcohol 4 times, an alcohol is changed every 30min；50% dehydration of alcohol 4 times, every
30min changes an alcohol；Sample is transferred on shaking table or shakes container, 75% dehydration of alcohol 4 every 8min afterwards
It is secondary, change an alcohol every 40min；95% dehydration of alcohol 4 times, an alcohol is changed every 40min；100% absolute alcohol dehydration 4
It is secondary, change an alcohol every 40min；
This step points for attention：
(1), change after 75% alcohol it can be found that the chlorophyll in Early filling stage seed fruit kind skin is gradually taken out by alcoholic extract
Come, close to the alcohol greening of bottom of bottle, therefore rocked using shaking table, to keep alcohol uniform, be smoothed out seed dehydration.
(2), this step can also be without using shaking table, but needs to shake container every 10min or so.To for several times finally
No chlorophyll is extracted out when changing alcohol, shows that seed dehydration is complete.
Conventional method often walks dewatering time and is generally 1~2h, and the step extends dewatering time compared with conventional method, together
When using shaking table make dehydration more smooth, and easily determine whether seed dehydration is completed.
（Three）, it is transparent：
After above-mentioned alcohol is suctioned out using imbibition rifle, clarifier is made using chloroform, addition volume ratio is alcohol：Chloroform=3：1
Solution left standstill 3h, it is evacuated to all material and sinks to the bottom；Solution is suctioned out using imbibition rifle afterwards, addition volume ratio is alcohol：Chlorine
Imitative=1：1 solution, 3h is stood, all material is evacuated to and sinks to the bottom；Reuse imbibition rifle to suction out solution, adding volume ratio is
Alcohol：Chloroform=1：3 solution, 3h is stood, all material is evacuated to and sinks to the bottom；Reuse imbibition rifle to suction out solution, add pure
Chloroform stands 3d, is evacuated to all material and sinks to the bottom；
This step points for attention：
（1）, chloroform will not make Material shrinkage be hardened, therefore use chloroform instead and make clarifier.But chloroform seepage velocity is slower, by
Being in the milk in wheat seed, later stage material itself is harder, water content is relatively low, therefore this step clearing time will more than conventional process time
It is long.
（2）, due to chloroform density it is larger, every time improve chloroform ratio when, material can all be floated to liquid level, so every time
Need to vacuumize to promote clarifier to enter inside material structure.
（3）, at the end of transparent step, it can be found that the grouting later stage（35d after such as pollinating）Material can also against light source
Printing opacity, show that the seed step is transparent good.
The step uses chloroform instead and makees clarifier, and significantly extends clearing time, can accomplish not make Material shrinkage be hardened
It is and transparent complete.
（Four）, waxdip：
After using imbibition rifle, the above-mentioned pure chloroform is suctioned out, addition volume ratio is chloroform：Paraffin=3：1 mixed liquor,
4h is incubated at 40 DEG C；Afterwards, after being suctioned out above-mentioned chloroform and paraffin mixed liquor using imbibition rifle, addition volume ratio is chloroform：Stone
Wax=1：1, it is incubated 4h at 40 DEG C；Afterwards, after above-mentioned chloroform and paraffin mixed liquor being suctioned out using imbibition rifle, add volume ratio
For chloroform：Paraffin=1：3 mixed liquor, 4h is incubated at 45 DEG C；For the material before 21d after pollination, using paraffin refined wax 55
3d is incubated at DEG C；For the later materials of 21d after pollination, 3d is incubated at 55 DEG C using paraffin refined wax, is changed once during waxdip pure
Paraffin；
This step points for attention：
（1）, wheat seed starch itself and protein content it is higher, it is especially even more so to the Grain Development later stage, cause to soak
Wax is very difficult.Therefore the waxdip time must be extended, and suitable temperature must be kept during waxdip, temperature is too high to be not only broken up
Structure inside seed, and higher than 60 DEG C when be unfavorable for preserve organization internal bioactive substance, influence SABC
As a result, also tissue can be made to be hardened, become fragile, also results in the overall loose, disintegration of seed, therefore, keep 55 DEG C of waxdips, make at paraffin
In slush state.
（2）, waxdip it is complete after, it can be found that material becomes micro- green or white translucent bowlder-like.
The step keeps relatively low waxdip temperature, but significantly extends the waxdip time, can make in seed histocyte
Complete waxdip, while can ensure that material is complete and does not destroy internal structure, moreover it is possible to preserve the bioactive substance of organization internal.
As shown in Fig. 2 be the inventive method and the wheat seed cross section comparison diagram after conventional method waxdip, A, B in figure
Be expressed as wheat spend rear 21d and 28d seed use this method waxdip after effect, C, D be respectively wheat spend rear 21d and
28d seed uses the effect after conventional method waxdip.It is as shown in Fig. 2 complete using the wheat seed waxdip after this method waxdip
Entirely, be translucent shape, and not having starch during section drops, and wheat seed waxdip is insufficient after using conventional method waxdip, cuts
Starch is had during piece to drop.
（Five）, embedding and section：
Used seed material is embedded according to a conventional method with carton or FFPE box, before 21d after pollination
Material directly can cut into slices on paraffin slicing machine according to a conventional method, must be used after material FFPE later 21d after pollination
Blade cuts out material cross-section, and then by wax stone back-off, 4 DEG C of softenings overnight, are then cut into slices again in DEPC water, above-mentioned institute
There is the section that seed material can be cut into 8 μm of thickness range.
This step adds DEPC water bating steps for Grain Development later stage material, makes the Grain Development later stage basic
Ripe seed can also cut out whole slices.
Through paraffin section made of the method after roasting piece, dewaxing and rehydration, it can be entered according to follow-up study experiment purpose
Enter ensuing experimental implementation.
As shown in figure 3, be the contrast picture of the wheat seed paraffin section cut out using the inventive method and conventional method,
Shown in figure：A, B, C be respectively wheat spend rear 21,28, the paraffin section that is cut out using this method of 35d seed, D, E, F difference
Spend rear 21 for wheat, 28, the paraffin section that is cut out using conventional method of 35d seed.
As shown in figure 3, the complete paraffin section of wheat seed development later stage can be cut out using the inventive method, and use
The paraffin section that conventional method is cut out is substantially all imperfect.
As shown in figure 4, the microphoto for wheat seed different times paraffin section after I2-KI is dyed, A, B in figure,
C, D be respectively wheat spend rear 14,21,28, the microphoto taken pictures immediately after I2-KI is dyed of 35d seed paraffin section, E,
F, G, H be respectively wheat spend rear 14,21,28, the microphoto taken pictures after I2-KI dyes 48h of 35d seed paraffin section.
Each period complete paraffin section after wheat seed is pollinated has been produced using the inventive method.If Fig. 4 is to use this method
The section of making is taken pictures immediately after I2-KI is dyed（A~D in figure）After dyeing 48h（E~H in figure）Take pictures to obtain micro-
Photo.From figure it will be clear that in wheat seed albuminous cell starch distribution（A~D in figure）And clearly cell
Structure（E~H in figure）.
Embodiment 2:
A kind of paraffin section preparation method for the close hard vegetable material of quality, comprise the following steps:
(1) it is, fixed:
After pollinating 7,14,21,28,35d each 5 of fresh wheat seed is taken respectively, vial is put into after crosscutting, is added
The paraformaldehyde fixers of 5ml 4%, are vacuumized using vacuum desiccator, all sink to container bottom to all seed materials, room temperature is solid
Determine 3h；
（Two）, dehydration：
Above-mentioned fixer is suctioned out using liquid-transfering gun, it is dense for 30%, 50%, 75%, 95% and 100% successively to sequentially add gradient
The alcohol of degree is dehydrated：30% dehydration of alcohol 3 times, an alcohol is changed every 25min；50% dehydration of alcohol 3 times, every
20min changes an alcohol；Sample is transferred on shaking table or shakes container, 75% dehydration of alcohol 5 every 10min afterwards
It is secondary, change an alcohol every 45min；95% dehydration of alcohol 5 times, an alcohol is changed every 45min；100% absolute alcohol dehydration 3
It is secondary, change an alcohol every 45min；
（Three）, it is transparent：
After above-mentioned alcohol is suctioned out using imbibition rifle, clarifier is made using chloroform, addition volume ratio is alcohol：Chloroform=3：1
Solution left standstill 4h, it is evacuated to all material and sinks to the bottom；Solution is suctioned out using imbibition rifle afterwards, addition volume ratio is alcohol：Chlorine
Imitative=1：1 solution, 3.5h is stood, all material is evacuated to and sinks to the bottom；Reuse imbibition rifle to suction out solution, add volume ratio
For alcohol：Chloroform=1：3 solution, 3.5h is stood, all material is evacuated to and sinks to the bottom；Reuse imbibition rifle to suction out solution, add
Enter pure chloroform and stand 2d, be evacuated to all material and sink to the bottom；
（Four）, waxdip：
After using imbibition rifle, the above-mentioned pure chloroform is suctioned out, addition volume ratio is chloroform：Paraffin=3：1 mixed liquor,
4.5h is incubated at 41 DEG C；Afterwards, after being suctioned out above-mentioned chloroform and paraffin mixed liquor using imbibition rifle, addition volume ratio is chloroform：
Paraffin=1：1, it is incubated 4.5h at 41 DEG C；Afterwards, after above-mentioned chloroform and paraffin mixed liquor being suctioned out using imbibition rifle, add body
Product ratio is chloroform：Paraffin=1：3 mixed liquor, 4.5h is incubated at 46 DEG C；For the material before 21d after pollination, pure stone is utilized
Wax is incubated 3.5d at 56 DEG C；For the later materials of 21d after pollination, it is incubated 4d at 56 DEG C using paraffin refined wax, during waxdip more
Change a paraffin refined wax；
（Five）, embedding and section：
Used seed material is embedded according to a conventional method with carton or FFPE box, before 21d after pollination
Material directly can cut into slices on paraffin slicing machine according to a conventional method, must be used after material FFPE later 21d after pollination
Blade cuts out material cross-section, and then by wax stone back-off, 5 DEG C of softenings overnight, are then cut into slices again in DEPC water, above-mentioned institute
There is the section that seed material can be cut into 20 μm of thickness range.
Embodiment 3:
A kind of paraffin section preparation method for the close hard vegetable material of quality, comprise the following steps:
(1) it is, fixed:
After pollinating 7,14,21,28,35d each 5 of fresh wheat seed is taken respectively, vial is put into after crosscutting, is added
The paraformaldehyde fixers of 5ml 4%, are vacuumized using vacuum desiccator, all sink to container bottom to all seed materials, room temperature is solid
Determine 5h；
（Two）, dehydration：
Above-mentioned fixer is suctioned out using liquid-transfering gun, it is dense for 30%, 50%, 75%, 95% and 100% successively to sequentially add gradient
The alcohol of degree is dehydrated：30% dehydration of alcohol 5 times, an alcohol is changed every 20min；50% dehydration of alcohol 5 times, every
25min changes an alcohol；Sample is transferred on shaking table or shakes container, 75% dehydration of alcohol 3 every 12min afterwards
It is secondary, change an alcohol every 50min；95% dehydration of alcohol 3 times, an alcohol is changed every 50min；100% absolute alcohol dehydration 5
It is secondary, change an alcohol every 50min；
（Three）, it is transparent：
After above-mentioned alcohol is suctioned out using imbibition rifle, clarifier is made using chloroform, addition volume ratio is alcohol：Chloroform=3：1
Solution left standstill 3.5h, it is evacuated to all material and sinks to the bottom；Solution is suctioned out using imbibition rifle afterwards, addition volume ratio is alcohol：
Chloroform=1：1 solution, 4h is stood, all material is evacuated to and sinks to the bottom；Reuse imbibition rifle to suction out solution, add volume ratio
For alcohol：Chloroform=1：3 solution, 4h is stood, all material is evacuated to and sinks to the bottom；Reuse imbibition rifle to suction out solution, add
Pure chloroform stands 2.5d, is evacuated to all material and sinks to the bottom；
（Four）, waxdip：
After using imbibition rifle, the above-mentioned pure chloroform is suctioned out, addition volume ratio is chloroform：Paraffin=3：1 mixed liquor,
5h is incubated at 42 DEG C；Afterwards, after being suctioned out above-mentioned chloroform and paraffin mixed liquor using imbibition rifle, addition volume ratio is chloroform：Stone
Wax=1：1, it is incubated 5h at 42 DEG C；Afterwards, after above-mentioned chloroform and paraffin mixed liquor being suctioned out using imbibition rifle, add volume ratio
For chloroform：Paraffin=1：3 mixed liquor, 5h is incubated at 47 DEG C；For the material before 21d after pollination, using paraffin refined wax 57
4d is incubated at DEG C；For the later materials of 21d after pollination, 5d is incubated at 57 DEG C using paraffin refined wax, is changed once during waxdip pure
Paraffin；
（Five）, embedding and section：
Used seed material is embedded according to a conventional method with carton or FFPE box, before 21d after pollination
Material directly can cut into slices on paraffin slicing machine according to a conventional method, must be used after material FFPE later 21d after pollination
Blade cuts out material cross-section, and then by wax stone back-off, 6 DEG C of softenings overnight, are then cut into slices again in DEPC water, above-mentioned institute
There is the section that seed material can be cut into 25 μm of thickness range.
Claims (1)
1. a kind of paraffin section preparation method for the close hard vegetable material of quality, it is characterised in that comprise the following steps:
(1) it is, fixed:
Take the plant seed material of different times after pollinating to carry out rip cutting or crosscutting, be put into the fixer in container, using true
Empty drier vacuumizes, and all sinks to container bottom to all seed materials, room temperature fixes 3~5h；
（Two）, dehydration：
Above-mentioned fixer is suctioned out using liquid-transfering gun, successively sequentially adds gradient as 30%, 50%, 75%, 95% and 100% concentration
Alcohol is dehydrated：30% dehydration of alcohol 3~5 times, an alcohol is changed every 20~30min；50% dehydration of alcohol 3~5 times, often
An alcohol is changed every 20~30min；Sample is transferred on shaking table or shakes container every 8~12min afterwards, 75%
Dehydration of alcohol 3~5 times, an alcohol is changed every 40~50min；95% dehydration of alcohol 3~5 times, changed every 40~50min
Alcohol；100% absolute alcohol is dehydrated 3~5 times, and an alcohol is changed every 40~50min；
（Three）, it is transparent：
After above-mentioned alcohol is suctioned out using imbibition rifle, clarifier is made using chloroform, addition volume ratio is alcohol：Chloroform=3：1 solution
3~4h is stood, all material is evacuated to and sinks to the bottom；Solution is suctioned out using imbibition rifle afterwards, addition volume ratio is alcohol：Chloroform
=1：1 solution, 3~4h is stood, all material is evacuated to and sinks to the bottom；Reuse imbibition rifle to suction out solution, adding volume ratio is
Alcohol：Chloroform=1：3 solution, 3~4h is stood, all material is evacuated to and sinks to the bottom；Reuse imbibition rifle to suction out solution, add
Enter pure chloroform and stand 2~3d, be evacuated to all material and sink to the bottom；
（Four）, waxdip：
After using imbibition rifle, the pure chloroform is suctioned out, addition volume ratio is chloroform：Paraffin=3：1 mixed liquor, at 40~42 DEG C
4~5h of lower insulation；Afterwards, after being suctioned out above-mentioned chloroform and paraffin mixed liquor using imbibition rifle, addition volume ratio is chloroform：Paraffin
=1：1,4~5h is incubated at 40~42 DEG C；Afterwards, after above-mentioned chloroform and paraffin mixed liquor being suctioned out using imbibition rifle, add body
Product ratio is chloroform：Paraffin=1：3 mixed liquor, 4~5h is incubated at 45~47 DEG C；For the material before 21d after pollination, profit
3~4d is incubated at 55~57 DEG C with paraffin refined wax；For the later materials of 21d after pollination, using paraffin refined wax in 55~57 DEG C of guarantors
3~5d of temperature, a paraffin refined wax is changed during waxdip；
（Five）, embedding and section：
Used seed material is embedded according to a conventional method with carton or FFPE box, for the material before 21d after pollination
Directly cut into slices on paraffin slicing machine, must be cut out after pollination after material FFPE later 21d using blade according to a conventional method
Material cross-section, then by wax stone back-off, 4~6 DEG C of softenings overnight, are then cut into slices again in DEPC water, above-mentioned all seeds
Material is cut into the section of 8~25 μm of thickness range.
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| CN106053177B (en) * | 2016-07-28 | 2018-10-23 | 江苏农林职业技术学院 | A kind of Slide processing for observing Tillandsia natomical leaf structure |
| CN106442531B (en) * | 2016-10-28 | 2019-02-26 | 广东省农业科学院环境园艺研究所 | A method of distinguishing Bowring cattleya flower quality |
| CN106525530B (en) * | 2016-11-04 | 2019-07-12 | 河南科技大学 | A kind of paraffin section method of trees stem tissue |
| CN108332989B (en) * | 2018-02-27 | 2020-12-29 | 江南大学 | Method for analyzing high-starch-content cereal grains and observing internal structure |
| CN110018033B (en) * | 2019-05-16 | 2021-08-10 | 鹤壁市人民医院 | Pathological specimen preparation device |
| CN110618008B (en) * | 2019-10-24 | 2021-11-05 | 中南大学湘雅三医院 | Pathological section exhibition piece drags for piece integer machine |
| CN112033723B (en) * | 2020-10-10 | 2024-03-29 | 北华大学 | Paraffin slicing method of tilia amurensis seeds |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102967493A (en) * | 2012-10-27 | 2013-03-13 | 山西农业大学 | Rapid paraffin sectioning method for plant tissue |
| CN103234797A (en) * | 2013-04-28 | 2013-08-07 | 中南大学湘雅三医院 | Dehydrating and embedding improvement method of human body or animal tissues |
| CN103305600A (en) * | 2013-01-25 | 2013-09-18 | 海尔施生物医药股份有限公司 | Kit for synchronously detecting related gene expression level of 14 antitumor drugs by using paraffin embedding biopsy sample, and detection method thereof |
| WO2015034178A1 (en) * | 2013-09-09 | 2015-03-12 | 국립암센터 | Blood cell aggregating agent for preparing paraffin block and method for preparing paraffin block by using same |
| JP2015114139A (en) * | 2013-12-10 | 2015-06-22 | 村角工業株式会社 | Tray for preparing paraffin block |
-
2016
- 2016-02-18 CN CN201610090674.4A patent/CN105699142B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102967493A (en) * | 2012-10-27 | 2013-03-13 | 山西农业大学 | Rapid paraffin sectioning method for plant tissue |
| CN103305600A (en) * | 2013-01-25 | 2013-09-18 | 海尔施生物医药股份有限公司 | Kit for synchronously detecting related gene expression level of 14 antitumor drugs by using paraffin embedding biopsy sample, and detection method thereof |
| CN103234797A (en) * | 2013-04-28 | 2013-08-07 | 中南大学湘雅三医院 | Dehydrating and embedding improvement method of human body or animal tissues |
| WO2015034178A1 (en) * | 2013-09-09 | 2015-03-12 | 국립암센터 | Blood cell aggregating agent for preparing paraffin block and method for preparing paraffin block by using same |
| JP2015114139A (en) * | 2013-12-10 | 2015-06-22 | 村角工業株式会社 | Tray for preparing paraffin block |
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