CN106525530A - Paraffin slicing method of tree stem tissue - Google Patents
Paraffin slicing method of tree stem tissue Download PDFInfo
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- CN106525530A CN106525530A CN201610965041.3A CN201610965041A CN106525530A CN 106525530 A CN106525530 A CN 106525530A CN 201610965041 A CN201610965041 A CN 201610965041A CN 106525530 A CN106525530 A CN 106525530A
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- 238000000034 method Methods 0.000 title claims abstract description 41
- 239000012188 paraffin wax Substances 0.000 title claims abstract description 28
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 178
- 235000019441 ethanol Nutrition 0.000 claims abstract description 60
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000004043 dyeing Methods 0.000 claims abstract description 14
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 13
- 230000018044 dehydration Effects 0.000 claims abstract description 11
- 238000006297 dehydration reaction Methods 0.000 claims abstract description 11
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 34
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 9
- 230000008520 organization Effects 0.000 claims description 7
- 239000012153 distilled water Substances 0.000 claims description 6
- 235000011187 glycerol Nutrition 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 5
- RZSYLLSAWYUBPE-UHFFFAOYSA-L Fast green FCF Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC(O)=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 RZSYLLSAWYUBPE-UHFFFAOYSA-L 0.000 claims description 4
- 238000005034 decoration Methods 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000001574 biopsy Methods 0.000 claims description 2
- 241000196324 Embryophyta Species 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract description 2
- 241001391944 Commicarpus scandens Species 0.000 abstract 2
- 238000004026 adhesive bonding Methods 0.000 abstract 1
- 235000019589 hardness Nutrition 0.000 abstract 1
- 238000003754 machining Methods 0.000 abstract 1
- 238000007789 sealing Methods 0.000 abstract 1
- 238000004018 waxing Methods 0.000 abstract 1
- 239000000463 material Substances 0.000 description 8
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 238000001000 micrograph Methods 0.000 description 6
- 239000001993 wax Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 241000968368 Picea meyeri Species 0.000 description 4
- 240000008289 Quercus suber Species 0.000 description 4
- 235000016977 Quercus suber Nutrition 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 241000411840 Larix gmelinii var. principis-rupprechtii Species 0.000 description 3
- 239000000853 adhesive Substances 0.000 description 3
- 230000001070 adhesive effect Effects 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 239000002023 wood Substances 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000009940 knitting Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 241000218652 Larix Species 0.000 description 1
- 241000078511 Microtome Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 241000219492 Quercus Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000002520 cambial effect Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 210000000020 growth cone Anatomy 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 210000004272 stretch cell Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000005418 vegetable material Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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
-
- 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 invention discloses a paraffin slicing method of tree stem tissue, and belongs to the technical field of plant microscopic structure slicing. The paraffin slicing method of the tree stem tissue comprises the operations of tissue fixing, primary softening, dehydration, transparency, waxing, embedding, secondary softening, slicing, slice folding, slice gluing, slicing roasting, dewaxing, rehydration, dyeing, slice sealing and the like, wherein the addition of softening treatment can solve the problem that phloem, a forming layer and xylem in the stem tissue are easy to break in the slicing process due to inconsistent hardnesses. The softening treatment comprises the steps of firstly after the tissue is fixed, adding the operation of using ethyl alcohol/glycerinum mixed liquor to soften the tissue; secondly after embedding and coarse machining, adding the operation of using warm water to soak so as to soften the tissue to be sliced. Experiments demonstrate that a softened tissue can lower probability that the stem tissue is easy to break during slicing, and improve slicing success rate.
Description
Technical field
The present invention relates to a kind of paraffin section method of trees stem tissue, belongs to plant microscopic tissue sections technology neck
Domain.
Background technology
Paraffin section is most widely used in histology conven tional tabletting techniques, is mainly used in the Appearance View of cell tissue
Examine, made by section permanent section load is made Jing after mounting, can preserve for a long time.
Trees stem tissue paraffin section technology is to study the important experiment skill of model for trees and Physio-ecological mechanism
Art.Trees stem tissue includes xylem, three part of forming layer and bast, and the hardness of this three is inconsistent.Xylem is usual
Tree material is hard, cell wall thickness；Cambial cell and tanycyte tissue are softer, and moisture is high, and cell membrane is thin；And bast
The cell membrane of tissue is relatively thin.
Conventional plant tissue paraffin section method includes fixing, is dehydrated the steps such as transparent, waxdip, embedding, section statining,
And the method is commonly available to the tissue that moisture is moderate, hardness is more consistent, such as blade, timber etc..Such as publication No.
A kind of paraffin section preparation method for the tight hard vegetable material of quality, bag disclosed in the patent of invention of CN105699142A
Include：1) it is fixed：By the rip cutting of plant seed material or it is crosscutting after be put in fixer, be evacuated to seed and sink to container bottom, room
Temperature fixes 3～5h；2) it is dehydrated：Material after fixation is sequentially placed in the ethanol of 30%, 50%, 75%, 95%, 100% concentration
Dehydration；3) it is transparent：Material after dehydration is sequentially placed into alcohol, chloroform volume ratio 3:1、1:1、1:3 mixed liquor and pure chloroform
Middle standing is transparent；4) waxdip：Material after transparent is sequentially placed into chloroform, paraffin volume ratio 3:1、1:1、1:3 mixed liquor and
Waxdip in paraffin refined wax；5) material after waxdip embeds according to a conventional method, cuts into slices.But, it is inconsistent for tissue hardness
Trees stem, the effect using conventional film-making are still not ideal enough, are mainly shown as material brittle or tissue quilt in slicing processes
Destruction.And the complex operation of conventional dicing method, takes longer.
Therefore, for the trees stem tissue that material is hard and hardness is inconsistent, needs are cut from methodology angle to paraffin
Chip technology is adjusted and perfect.
The content of the invention
It is an object of the invention to provide a kind of paraffin section method of trees stem tissue, to solve bast in stem tissue
Portion, forming layer and xylem three because hardness is inconsistent cause slicing processes in organize frangible technical problem.
In order to realize object above, the technical solution adopted in the present invention is：
A kind of paraffin section method of trees stem tissue, comprises the following steps：
(1) tissue is fixed：
Trees stem tissue is dipped in fixer fixed；
(2) once soften：
Tissue after fixation is dipped in into volume ratio 1:Soften in 1 70% ethanol and the bating liquor of glycerine；
(3) tissue dewatering：
Tissue after softening is dipped in the ethanol that concentration gradient is 35%, 70%, 90%, 95%, 100% successively and is taken off
Water, each gradient at least process 40min；
(4) transparency of organization：
Tissue after dehydration is first dipped in into volume ratio 1:In 1 100% ethanol and the mixed liquor of dimethylbenzene, then it is dipped in diformazan
It is transparent in benzene；
(5) waxdip and embedding：
Tissue after will be transparent is dipped in waxdip in atoleine, embeds after waxdip；
(6) secondary softening：
Tissue after embedding is dipped in water half an hour to soften to one week；
(7) section, exhibition piece, bonding die and roasting piece；
(8) dewaxing and rehydration：
After biopsy tissues are with dimethylbenzene dewaxing treatment, then successively with the ethanol that concentration gradient is 100%, 95%, 70% and
Water rehydration；
(9) dyeing and mounting：
Using sarranine-fast green decoration method dyeing, mounting.
In step (1), stem tissue takes from (1.3m or so) at tree breast-height diameter, and tissue size is 1.5～2.5mm of diameter, long
15～20mm of degree.Xylem region of the stem tissue comprising bast, forming layer and 3～5 years.
It is put in fixer rapidly after stem tissue sampling in step (1), is evacuated to tissue and is sink to sample bottle bottom completely
Portion, it is stored refrigerated at 4～6 DEG C.Fixer is 9 by volume ratio:0.5:0.5 70% ethanol, formaldehyde and acetic acid composition.Fixer
The volume ratio of consumption and stem tissue should be greater than 20:1, so that stem tissue is completely attached to fixer.Regular time is 5
～8d (one week or so).
The time softened in step (2), depending on tissue soft or hard degree, general 1～4 week, can use pocket knife to tissue xylem
Till easily cutting off.The consumption of bating liquor should be greater than 20 with the volume ratio of stem tissue:1.
The concrete operations of tissue dewatering in step (3) are：
1) 1h is kept in 35% ethanol；
2) 1h is kept in 70% ethanol；
3) 50min is kept in 90% ethanol；
4) repeat step 3) middle operation 1 time；
5) 50min is kept in 95% ethanol；
6) the holding 40min in 100% ethanol (i.e. absolute ethyl alcohol)；
7) repeat step 6) middle operation 1 time.Step 1)～7) in operation carry out at room temperature.
The concrete operations of transparency of organization in step (4) are：
1) in volume ratio 1:40min is kept in 1 100% ethanol and the mixed liquor of dimethylbenzene；
2) 40min is kept in dimethylbenzene；
3) repeat step 2) middle operation 1 time.Step 1)～3) in operation carry out at room temperature.
The concrete operations of waxdip in step (5) are：
1) 20min is kept in atoleine；
2) 30min is kept in atoleine；
3) repeat step 2) middle operation 1 time.Soft wax of the paraffin using 58～60 DEG C of fusing point.
In step (5) waxdip as adopt pure manual operations, before paraffin refined wax waxdip, can prior to 37～40 DEG C at adopt volume
Than 1:1 dimethylbenzene and the mixed liquor 48～72h of waxdip of paraffin.
The stem tissue after embedding is repaiied block first before softening in step (6) to be rectangle, rough cut appears stem tissue；
Tissue after rough cut is dipped in water, softens stem tissue, treat that tissue slightly expands softening and stops.The time of softening regards group
Depending on knitting soft durometer, temperature is 30～35 DEG C, and normal temperature also may be used.
Section in step (7) can adopt biologic slice machine, cycle type, full rotation type.During section, stem tissue should hang down
Directly in edge of a knife direction, 8～12 μm of thickness.After section, piece is opened up in 40～45 DEG C of warm water.Bonding die adopts volume ratio 1:1 egg
Make adhesive with the mixture of glycerine clearly.During bonding die, first by adhesive uniform application on slide, to strengthen section and carry glass
The caking property of piece, prevents from coming off in the operations such as follow-up dewaxing, rehydration, dyeing.After bonding die, slide is baked at 35～39 DEG C
24～36h of piece.Slide after roasting piece can deposit 2～3d.
The concrete operations of dewaxing in step (8) are：
1) 5～10min of holding in dimethylbenzene；
2) repeat step 1) middle operation 3 times.Before dewaxing, slide is first heated at 45～65 DEG C 3～5min；
The concrete operations of rehydration in step (8) are：
1) 20s is kept in 100% ethanol；
2) 20s is kept in 95% ethanol；
3) 20s is kept in 70% ethanol；
4) 20s is kept in distilled water.
The concrete operations of dyeing in step (9) are：
1) 15～20min of holding in the 1% sarranine aqueous solution；
2) 10s is kept in distilled water；
3) 10s is kept in 70% ethanol；
4) repeat step 3) middle operation 3 times；
5) 30s is kept in 0.5% 95% fast green ethanol solution；
6) 10s is kept in 95% ethanol；
7) repeat step 6) middle operation 1 time；
8) 10s is kept in absolute ethyl alcohol；
9) repeat step 8) middle operation 1 time.
In step (9), mounting adopts neutral gum.
Beneficial effects of the present invention：
In the present invention paraffin section method of trees stem tissue include that tissue is fixed, once soften, dehydration, transparent, leaching
The operation such as wax, embedding, secondary softening, section, exhibition piece, bonding die, roasting piece, dewaxing, rehydration, dyeing and mounting, wherein increase softening
Process can solve the problem that in stem tissue that bast, forming layer and xylem three cause group in slicing processes because hardness is inconsistent
The problems such as knitting frangible.Sofening treatment includes that two steps are operated, and one is, after tissue is fixed, to increase and softened with ethanol/glycerine mixed liquor
The operation of tissue, two is the operation that increase is soaked with warm water, to soften tissue to be cut after embedding rough cut.Test is proved, soft
Changing tissue can reduce the frangible probability of stem tissue in section, improve section success rate.
The present invention reached simplified operation, has shortened the good of time by the time for adjusting dehydration, transparent and waxdip is processed
Effect.By taking dehydration as an example, due to 35% ethanol used in organization softening, dehydration initial concentration is from the beginning of 35% concentration of alcohol；By
It is relatively low in stem tissue water content, it is appropriate in dehydrating operations to reduce ethanol series, you can to reach the effect of moisture displacement；And group
Knit that itself is more crisp, and high concentration ethanol can aggravate tissue and become fragile, thus when being dehydrated with absolute ethyl alcohol the time need to shorten, with
40min is advisable.On the premise of dicing effect is ensured, the inventive method can simplify step, shorten the time, cut into slices with routine paraffin wax
Compare, shorten within 3 days 1 day, be greatly improved microsection manufacture efficiency, while reducing the use of various reagents, can not only save
Cost, can also reduce waste liquid to environment, the injury of operating personnel.Meanwhile, the perfect dyeing side for being suitable to stem tissue of the present invention
Case, can distinguish bast, three regions of forming layer and xylem with sarranine-fast green decoration method dyeing so that tissue shows clear
Clear, each position discrimination is high, can strengthen section and observing effect.The method contributes to abundant paraffin section and makes theoretical, is one
Kind is worthy to be popularized, effective flaking method.
Description of the drawings
Fig. 1 is the cross section micrograph of Picea meyeri stem tissue in embodiment 1；
Fig. 2 is the cross section micrograph of Larix principis-rupprechtii stem tissue in embodiment 2；
Fig. 3 is the cross section micrograph of cork oak stem tissue in embodiment 3.
Specific embodiment
Following embodiments are only described in further detail to the present invention, but do not constitute any limitation of the invention.
Embodiment 1
In the present embodiment, the paraffin section method of trees stem tissue, comprises the following steps that：
(1) stem tissue sampling：
In the wild, with micro- growth cone (Trephor, forming layer sampler) in Picea meyeri (Picea meyeri Rebd.Et
Wils) at the diameter of a cross-section of a tree trunk 1.3 meters above the ground (1.3m or so) collection stem tissue sample, tissue size be diameter 2mm, length 18mm, comprising bast, shape
The xylem region of stratification and 4 years；
(2) tissue is fixed：
After sampling, stem tissue is put into rapidly and fills omnipotent fixer (70% ethanol of volume ratio:Formaldehyde:Acetic acid=9:
0.5:0.5) in 7mL penicillin bottles, (consumption of fixer is more than 20 with the volume ratio of stem tissue:1), taken out very with syringe
It is empty so that stem tissue is sink to bottom of bottle completely, is then placed in stored refrigerated 1 week in 5 DEG C of incubators, note keeping during preservation
The erectility of sample bottle；
(3) once soften：
Stem tissue after fixation is put into into 70% ethanol of volume ratio:Glycerine=1:Soften 1 week in 1 bating liquor, soften
The consumption of liquid is more than 20 with the volume ratio of stem tissue:1 (this step can long-term storage, but need to prevent alcohol from volatilizing)；
(4) tissue dewatering：
The stem tissue after softening is taken, under room temperature, is 35%, 70%, 90%, 95%, 100% with concentration gradient successively
Ethanol dehydration；Concrete operations are as follows：
1) 1h is kept in 35% ethanol；
2) 1h is kept in 70% ethanol；
3) 50min is kept in 90% ethanol；
4) repeat step 3) middle operation 1 time；
5) 50min is kept in 95% ethanol；
6) the holding 40min in 100% ethanol (i.e. absolute ethyl alcohol)；
7) repeat step 6) middle operation 1 time；
(5) transparency of organization：
The stem tissue after dehydration is taken, under room temperature, is carried out with 100% ethanol/dimethylbenzene mixed liquor, dimethylbenzene successively transparent
Process；Concrete operations are as follows：
1) in volume ratio 1:40min is kept in 1 100% ethanol and the mixed liquor of dimethylbenzene；
2) 40min is kept in dimethylbenzene；
3) repeat step 2) middle operation 1 time；
(6) waxdip and embedding：
At 65 DEG C be in advance 58～60 DEG C paraffin melting by fusing point, take it is transparent after stem tissue, with paraffin waxdip,
Embedding；Concrete operations are as follows：
1) 20min is kept in atoleine；
2) 30min is kept in atoleine；
3) repeat step 2) middle operation 1 time；
4) paraffin of third time is put in embedding grinding tool together with stem tissue, rapid adjustment is organized in the position in paraffin
Put, solidification embedding is completed in cold bench；
(7) secondary softening：
The stem tissue after embedding is taken, rectangle of the block in rule is repaiied, it is thick using 2235 cycle type slicers of Leica RM
Cutting, sets 8 μm of slice thickness, makes stem tissue Essential Emerging；Under room temperature, the tissue of rough cut is dipped in into 2h in water, stem is made
Stem organization softens, and stops when tissue slightly expands；
(8) section, exhibition piece, bonding die and roasting piece：
Using 2235 cycle type microtomes of Leica RM, 8 μm of slice thickness is set, during section, make stem tissue hang down
Directly in edge of a knife direction, put forth effort area to reduce；The wax band for cutting, is removed and is put in the tank of 40 DEG C of water temperature with tweezers and open up piece；
After 4min, with good adhesive (the volume ratio egg of advance uniform application:Glycerine=1:1) slide, wax band bonding die is sprawled；
Excessive moisture is absorbed with filter paper, slide is put in 37 DEG C of incubator and is baked piece 24h；
(9) dewaxing and rehydration：
Before dewaxing, slide is put in 65 DEG C of baking piece machines and heats 3min；Dewaxing, the concrete operations of rehydration are as follows：
1) 5min is kept in dimethylbenzene；
2) repeat step 1) middle operation 3 times；
3), after dewaxing, 20s is kept in 100% ethanol；
4) 20s is kept in 95% ethanol；
5) 20s is kept in 70% ethanol；
6) 20s is kept in distilled water；
(10) dyeing and mounting：
Dewaxing, the slide after rehydration are taken, using sarranine-fast green decoration method dyeing, concrete operations are as follows：
1) 15min is kept in the 1% sarranine aqueous solution；
2) 10s is kept in distilled water；
3) 10s is kept in 70% ethanol；
4) repeat step 3) middle operation 3 times；
5) 30s is kept in 0.5% 95% fast green ethanol solution；
6) 10s is kept in 95% ethanol；
7) repeat step 6) middle operation 1 time；
8) 10s is kept in absolute ethyl alcohol；
9) repeat step 8) middle operation 1 time；
10) after dyeing, with neutral gum mounting.
Above-mentioned Picea meyeri (hardness of wood is I grade) the cross section micrograph of stem tissue for preparing is shown in Fig. 1.In figure, 1 is
Bast, 2 is forming layer, and 3 is xylem, and in figure, scale is 100 μm.
Embodiment 2
The paraffin section method of trees stem tissue in the present embodiment, except collection Larix principis-rupprechtii (Larix in step (1)
Principis-rupprechtii Mayr.) stem tissue at the diameter of a cross-section of a tree trunk 1.3 meters above the ground, the embedded block after block will be repaiied in step (7) and is put into 30 DEG C
Soften in thermostat water bath outside 2d, other operations are with embodiment 1.
Above-mentioned Larix principis-rupprechtii (hardness of wood is III grade) the cross section micrograph of stem tissue for preparing is shown in Fig. 1.
In figure, 1 is bast, and 2 is forming layer, and 3 is xylem, and in figure, scale is 100 μm.
Embodiment 3
The paraffin section method of trees stem tissue in the present embodiment, except collection cork oak (Quercus in step (1)
Variabilis Bl.) stem tissue (as cork oak bark is thicker, need to suitably divest outer bark) at the diameter of a cross-section of a tree trunk 1.3 meters above the ground, in step (7)
To repair during the embedded block after block is put into 30 DEG C of thermostat water baths and soften outside 7d, other operations are with embodiment 1.
Above-mentioned cork oak (hardness of wood is IV～V grade) the cross section micrograph of stem tissue for preparing is shown in Fig. 1.
In figure, 1 is bast, and 2 is forming layer, and 3 is xylem, and in figure, scale is 100 μm.
Explanation：In text, " % " related to ethanol all refers to volume fraction, 1% sarranine and 0.5% 95% fast green ethanol
" % " in solution all refers to mass fraction.
Claims (10)
1. a kind of paraffin section method of trees stem tissue, it is characterised in that：Comprise the following steps：
(1) tissue is fixed：
Trees stem tissue is dipped in fixer fixed；
(2) once soften：
Tissue after fixation is dipped in into volume ratio 1:Soften in 1 70% ethanol and the bating liquor of glycerine；
(3) tissue dewatering：
Tissue after softening is dipped in the ethanol that concentration gradient is 35%, 70%, 90%, 95%, 100% successively and is dehydrated, often
Individual gradient at least processes 40min；
(4) transparency of organization：
Tissue after dehydration is first dipped in into volume ratio 1:In 1 100% ethanol and the mixed liquor of dimethylbenzene, then it is dipped in dimethylbenzene
It is transparent；
(5) waxdip and embedding：
Tissue after will be transparent is dipped in waxdip in atoleine, embeds after waxdip；
(6) secondary softening：
Tissue after embedding is dipped in water half an hour to soften to one week；
(7) section, exhibition piece, bonding die and roasting piece；
(8) dewaxing and rehydration：
It is after biopsy tissues are with dimethylbenzene dewaxing treatment then multiple with the second alcohol and water that concentration gradient is 100%, 95%, 70% successively
Water；
(9) dyeing and mounting：
Using sarranine-fast green decoration method dyeing, mounting.
2. method according to claim 1, it is characterised in that：In step (1), stem tissue is taken from tree breast-height diameter, tissue
Size is 1.5～2.5mm of diameter, 15～20mm of length.
3. method according to claim 1, it is characterised in that：In step (1), fixer is 9 by volume ratio:0.5:0.5
70% ethanol, formaldehyde and acetic acid composition.
4. method according to claim 1, it is characterised in that：The time softened in step (2) is 1～4 week.
5. method according to claim 1, it is characterised in that：The concrete operations of tissue dewatering in step (3) are：
1) 1h is kept in 35% ethanol；
2) 1h is kept in 70% ethanol；
3) 50min is kept in 90% ethanol；
4) repeat step 3) middle operation 1 time；
5) 50min is kept in 95% ethanol；
6) 40min is kept in 100% ethanol；
7) repeat step 6) middle operation 1 time.
6. method according to claim 1, it is characterised in that：The concrete operations of transparency of organization in step (4) are：
1) in volume ratio 1:40min is kept in 1 100% ethanol and the mixed liquor of dimethylbenzene；
2) 40min is kept in dimethylbenzene；
3) repeat step 2) middle operation 1 time.
7. method according to claim 1, it is characterised in that：The concrete operations of waxdip in step (5) are：
1) 20min is kept in atoleine；
2) 30min is kept in atoleine；
3) repeat step 2) middle operation 1 time.
8. method according to claim 1, it is characterised in that：The concrete operations of dewaxing in step (8) are：
1) 5～10min of holding in dimethylbenzene；
2) repeat step 1) middle operation 3 times.
9. method according to claim 1, it is characterised in that：The concrete operations of rehydration in step (8) are：
1) 20s is kept in 100% ethanol；
2) 20s is kept in 95% ethanol；
3) 20s is kept in 70% ethanol；
4) 20s is kept in distilled water.
10. method according to claim 1, it is characterised in that：The concrete operations of dyeing in step (9) are：
1) 15～20min of holding in the 1% sarranine aqueous solution；
2) 10s is kept in distilled water；
3) 10s is kept in 70% ethanol；
4) repeat step 3) middle operation 3 times；
5) 30s is kept in 0.5% 95% fast green ethanol solution；
6) 10s is kept in 95% ethanol；
7) repeat step 6) middle operation 1 time；
8) 10s is kept in absolute ethyl alcohol；
9) repeat step 8) middle operation 1 time.
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