CN106525530B - A kind of paraffin section method of trees stem tissue - Google Patents
A kind of paraffin section method of trees stem tissue Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 31
- 239000012188 paraffin wax Substances 0.000 title claims abstract description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 123
- 235000019441 ethanol Nutrition 0.000 claims abstract description 57
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 19
- 238000004043 dyeing Methods 0.000 claims abstract description 13
- 229960004756 ethanol Drugs 0.000 claims description 52
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 29
- 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
- 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 8
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 6
- 230000008520 organization Effects 0.000 claims description 6
- 238000005034 decoration Methods 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 238000001574 biopsy Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 230000018044 dehydration Effects 0.000 abstract description 5
- 238000006297 dehydration reaction Methods 0.000 abstract description 5
- 238000012360 testing method Methods 0.000 abstract description 2
- 239000011521 glass Substances 0.000 description 9
- 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
- 241000196324 Embryophyta Species 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
- 210000002421 cell wall Anatomy 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000002023 wood Substances 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 241000218652 Larix 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
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- QUBBAXISAHIDNM-UHFFFAOYSA-N ethyldimethylbenzene Natural products CCC1=CC=CC(C)=C1C QUBBAXISAHIDNM-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000000020 growth cone Anatomy 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000009940 knitting Methods 0.000 description 1
- 230000007774 longterm Effects 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
- 238000004321 preservation Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 210000004272 stretch cell Anatomy 0.000 description 1
- 239000005418 vegetable material Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
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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
-
- 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
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- Life Sciences & Earth Sciences (AREA)
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- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
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- Engineering & Computer Science (AREA)
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Abstract
The invention discloses a kind of paraffin section methods of trees stem tissue, belong to plant microscopic tissue sections technical field.Should include that tissue be fixed, once softening, dehydration, transparent, waxdip, embedding, secondary softening, slice, exhibition piece, bonding die, roasting piece, dewaxing, rehydration, dyeing and mounting etc. operate, wherein increase sofening treatment be able to solve bast in stem tissue, forming layer and xylem three because hardness is inconsistent lead to problems such as to organize in slicing processes it is frangible.Sofening treatment is operated including two steps, first is that increase after tissue is fixed and use the operation of ethyl alcohol/glycerol mixed liquor softening tissue, second is that after embedding rough cut, the operation that increase is impregnated with warm water, to soften tissue to be cut.Test proves that softening tissue can reduce the probability that stem tissue is frangible in slice, improves slice success rate.
Description
Technical field
The present invention relates to a kind of paraffin section methods of trees stem tissue, belong to plant microscopic tissue sections technology neck
Domain.
Background technique
Paraffin section is most widely used in histology conven tional tabletting techniques, is mainly used for the Appearance View of cell tissue
It examines, permanent slice load is made in manufactured slice after mounting, can long-term preservation.
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, forming layer and bast three parts, the hardness of this three is inconsistent.Xylem is usual
Material is hard, cell wall thickness；Cambial cell and tanycyte tissue are softer, and moisture content is high, and cell wall is thin；And bast
The cell wall of tissue is relatively thin.
Conventional plant tissue paraffin section method include it is fixed, be dehydrated transparent, waxdip, embedding, slice dyeing,
And this method is commonly available to moisture content is moderate, hardness is more consistent tissue, such as blade, timber.Such as publication No.
A kind of paraffin section production method for the close hard vegetable material of quality, packet disclosed in the patent of invention of CN105699142A
Include: 1) it is fixed: by plant seed material it is longitudinal sectional or crosscutting after be put into fixer, be evacuated to seed and sink to container bottom, room
Temperature fixes 3~5h；2) be dehydrated: the material after fixed is sequentially placed into the ethyl alcohol of 30%, 50%, 75%, 95%, 100% concentration
Dehydration；3) transparent: dewatered material is sequentially placed into the mixed liquor and pure chloroform of alcohol, chloroform volume ratio 3:1,1:1,1:3
Middle standing is transparent；4) waxdip: the material after transparent be 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 is embedded according to a conventional method, is sliced.But it is inconsistent for tissue hardness
Trees stem, the effect using conventional film-making is still not ideal enough, is mainly shown as material brittle or tissue quilt in slicing processes
It destroys.And conventional dicing method is complicated for operation, takes a long time.
Therefore, trees stem tissue hard for material and inconsistent hardness needs to cut paraffin from methodology angle
Chip technology is adjusted and perfect.
Summary of the invention
The object of the present invention is to provide a kind of paraffin section methods of trees stem tissue, to solve bast in stem tissue
Portion, forming layer and xylem three cause to organize frangible technical problem in slicing processes because hardness is inconsistent.
In order to achieve the goal above, the technical scheme adopted by the invention is that:
A kind of paraffin section method of trees stem tissue, comprising the following steps:
(1) tissue is fixed:
Trees stem tissue is dipped in fixer fixed；
(2) primary softening:
Tissue after fixation is dipped in 70% ethyl alcohol of volume ratio 1:1 and the bating liquor of glycerol and is softened；
(3) tissue dewatering:
Tissue after softening is successively dipped in the ethyl alcohol that concentration gradient is 35%, 70%, 90%, 95%, 100% and is taken off
Water, each gradient at least handle 40min；
(4) transparency of organization:
Dewatered tissue is first dipped in 100% ethyl alcohol of volume ratio 1:1 and the mixed liquor of dimethylbenzene, then 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) it is sliced, opens up piece, bonding die and roasting piece；
(8) dewaxing and rehydration:
After biopsy tissues dimethylbenzene dewaxing treatment, then successively with concentration gradient be 100%, 95%, 70% ethyl alcohol and
Water rehydration；
(9) dyeing and mounting:
Using the fast green decoration method dyeing of sarranine-, mounting.
Stem tissue is derived from tree breast-height diameter (1.3m or so) in step (1), and tissue size is 1.5~2.5mm of diameter, long
Spend 15~20mm.Stem tissue includes bast, forming layer and 3~5 years xylem regions.
It is put into 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 made of 70% ethyl alcohol, formaldehyde and the acetic acid that volume ratio is 9:0.5:0.5.Fixer
Dosage and the volume ratio of stem tissue should be greater than 20:1 so that stem tissue and fixer completely attach to.Regular time is 5
~8d (one week or so).
The time softened in step (2) depending on the soft or hard degree of tissue depending on, general 1~4 week, until organizing xylem that can use pocket knife
Easily until cutting.The dosage of bating liquor and the volume ratio of stem tissue should be greater than 20:1.
The concrete operations of tissue dewatering in step (3) are as follows:
1) 1h is kept in 35% ethyl alcohol；
2) 1h is kept in 70% ethyl alcohol；
3) 50min is kept in 90% ethyl alcohol；
4) operation 1 time in step 3) is repeated；
5) 50min is kept in 95% ethyl alcohol；
6) 40min is kept in 100% ethyl alcohol (i.e. dehydrated alcohol)；
7) operation 1 time in step 6) is repeated.Step 1)~7) in operation carry out at room temperature.
The concrete operations of transparency of organization in step (4) are as follows:
1) 40min is kept in the mixed liquor of 100% ethyl alcohol of volume ratio 1:1 and dimethylbenzene；
2) 40min is kept in dimethylbenzene；
3) operation 1 time in step 2) is repeated.Step 1)~3) in operation carry out at room temperature.
The concrete operations of waxdip in step (5) are as follows:
1) 20min is kept in atoleine；
2) 30min is kept in atoleine；
3) operation 1 time in step 2) is repeated.Paraffin uses 58~60 DEG C of fusing point of soft wax.
In step (5) waxdip as use pure manual operations, before paraffin refined wax waxdip, can prior to 37~40 DEG C at use volume
48~72h of mixed liquor waxdip of dimethylbenzene and paraffin than 1:1.
The stem tissue after embedding is first repaired block before softening in step (6) to be rectangle, rough cut appears stem tissue；
Tissue after rough cut is dipped in water, stem tissue is softened, wait organize slightly expansion softening to stop.The time view group of softening
Depending on knitting hardness, temperature is 30~35 DEG C, and room temperature can also.
Biologic slice machine, cycle type, full rotation type can be used in slice in step (7).When slice, stem tissue should hang down
Directly in edge of a knife direction, 8~12 μm of thickness.After slice, piece is opened up in 40~45 DEG C of warm water.Bonding die uses the egg of volume ratio 1:1
Make adhesive with the mixture of glycerol clearly.When bonding die, first adhesive is uniformly applied on glass slide, with enhancing slice and load glass
The caking property of piece prevents from falling off in the operation such as subsequent dewaxing, rehydration, dyeing.After bonding die, glass slide is baked at 35~39 DEG C
24~36h of piece.Glass slide after roasting piece can store 2~3d.
The concrete operations to dewax in step (8) are as follows:
1) 5~10min is kept in dimethylbenzene；
2) operation 3 times in step 1) are repeated.Before dewaxing, glass slide is first heated to 3~5min at 45~65 DEG C；
The concrete operations of rehydration in step (8) are as follows:
1) 20s is kept in 100% ethyl alcohol；
2) 20s is kept in 95% ethyl alcohol；
3) 20s is kept in 70% ethyl alcohol；
4) 20s is kept in distilled water.
The concrete operations dyed in step (9) are as follows:
1) 15~20min is kept in 1% sarranine aqueous solution；
2) 10s is kept in distilled water；
3) 10s is kept in 70% ethyl alcohol；
4) operation 3 times in step 3) are repeated；
5) 30s is kept in 0.5% 95% fast green ethanol solution；
6) 10s is kept in 95% ethyl alcohol；
7) operation 1 time in step 6) is repeated；
8) 10s is kept in dehydrated alcohol；
9) operation 1 time in step 8) is repeated.
Mounting uses neutral gum in step (9).
Beneficial effects of the present invention:
The paraffin section method of trees stem tissue includes that tissue is fixed, once softens, be dehydrated, be transparent, soaked in the present invention
The operations such as wax, embedding, secondary softening, slice, exhibition piece, bonding die, roasting piece, dewaxing, rehydration, dyeing and mounting, wherein increasing softening
Processing, which is able to solve bast in stem tissue, forming layer and xylem three, leads to group in slicing processes because hardness is inconsistent
Knit the problems such as frangible.Sofening treatment is operated including two steps, first is that increasing after tissue is fixed with ethyl alcohol/glycerol mixed liquor softening
The operation of tissue, second is that after embedding rough cut, the operation that increase is impregnated with warm water, to soften tissue to be cut.Test proves, soft
The probability that stem tissue is frangible in slice can be reduced by changing tissue, improve slice success rate.
The present invention has reached simplified operation, has shortened the good of time by adjusting the time of dehydration, the processing of transparent and waxdip
Effect.By taking dehydration as an example, due to using 35% ethyl alcohol in organization softening, initial concentration is dehydrated since 35% concentration of alcohol；By
It is lower in stem tissue water content, ethyl alcohol series is suitably reduced in dehydrating operations, that is, can 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 dehydrated alcohol the time need to shorten, with
40min is advisable.Under the premise of guaranteeing dicing effect, the method for the present invention can simplify step, shorten the time, be sliced with routine paraffin wax
It compares, 3 days is shorten to 1 day, microsection manufacture efficiency is greatly improved, while reducing the use of various reagents, can not only be saved
Cost can also reduce injury of the waste liquid to environment, operator.Meanwhile the perfect dyeing side suitable for stem tissue of the present invention
Case can distinguish three bast, forming layer and xylem regions with the fast green decoration method dyeing of sarranine-, so that tissue display is clear
Clear, each position discrimination is high, can enhance slice and observing effect.This method facilitates abundant paraffin section production theory, is one
It kind is worthy to be popularized, effective flaking method.
Detailed description of the invention
Fig. 1 is the micrograph in Picea meyeri stem tissue cross section in embodiment 1；
Fig. 2 is the micrograph in Larix principis-rupprechtii stem tissue cross section in embodiment 2；
Fig. 3 is the micrograph in cork oak stem tissue cross section in embodiment 3.
Specific embodiment
Only invention is further described in detail for following embodiments, but does not constitute any limitation of the invention.
Embodiment 1
The paraffin section method of trees stem tissue in the present embodiment, the specific steps are as follows:
(1) stem tissue samples:
In field, with micro- growth cone (Trephor, forming layer sampler) in Picea meyeri (Picea meyeri Rebd.Et
Wils) (1.3m or so) acquires stem tissue sample at the diameter of a cross-section of a tree trunk 1.3 meters above the ground, and it includes bast, shape that tissue size, which is diameter 2mm, length 18mm,
Stratification and 4 years xylem regions；
(2) tissue is fixed:
After sampling, stem tissue is put into rapidly fill omnipotent fixer (70% ethyl alcohol of volume ratio: formaldehyde: acetic acid=9:
In 7mL penicillin bottle 0.5:0.5) (dosage of fixer and the volume ratio of stem tissue are greater than 20:1), taken out with syringe true
Sky is then placed in 5 DEG C of incubators stored refrigerated 1 week so that stem tissue is sink to bottom of bottle completely, pays attention to keeping during saving
The upright state of sample bottle；
(3) primary softening:
Stem tissue after fixation is put into 70% ethyl alcohol of volume ratio: being softened 1 week in glycerol=1:1 bating liquor, is softened
The dosage of liquid and the volume ratio of stem tissue are greater than 20:1 (this step can be stored for a long time, but need to prevent alcohol from volatilizing)；
(4) tissue dewatering:
Stem tissue after taking softening is successively at room temperature 35%, 70%, 90%, 95%, 100% with concentration gradient
Ethanol dehydration；Concrete operations are as follows:
1) 1h is kept in 35% ethyl alcohol；
2) 1h is kept in 70% ethyl alcohol；
3) 50min is kept in 90% ethyl alcohol；
4) operation 1 time in step 3) is repeated；
5) 50min is kept in 95% ethyl alcohol；
6) 40min is kept in 100% ethyl alcohol (i.e. dehydrated alcohol)；
7) operation 1 time in step 6) is repeated；
(5) transparency of organization:
Dewatered stem tissue is taken, at room temperature, is successively carried out with 100% ethyl alcohol/dimethylbenzene mixed liquor, dimethylbenzene transparent
Processing；Concrete operations are as follows:
1) 40min is kept in the mixed liquor of 100% ethyl alcohol of volume ratio 1:1 and dimethylbenzene；
2) 40min is kept in dimethylbenzene；
3) operation 1 time in step 2) is repeated；
(6) waxdip and embedding:
Be in advance 58~60 DEG C paraffin melting by fusing point at 65 DEG C, 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) operation 1 time in step 2) is repeated；
4) paraffin of third time is put into embedding grinding tool together with stem tissue, rapid adjustment group is woven in the position in paraffin
It sets, solidification embedding is completed in cold bench；
(7) secondary softening:
Stem tissue after taking embedding repairs the rectangle that block is in rule, thick using 2235 cycle type slicer of Leica RM
Cutting sets 8 μm of slice thickness, makes stem tissue Essential Emerging；At room temperature, the tissue of rough cut is dipped in 2h in water, makes stem
Stem organization's softening, stopping when organizing slightly to expand；
(8) it is sliced, opens up piece, bonding die and roasting piece:
Using 2235 cycle type microtome of Leica RM, 8 μm of slice thickness are set, when slice, stem tissue is made to hang down
Directly in edge of a knife direction, put forth effort area to reduce；The wax band cut is removed and is put into 40 DEG C of water temperature of sink with tweezers and opens up piece；
After 4min, with the glass slide for wiping adhesive (volume ratio egg white: glycerol=1:1) is uniformly applied in advance, wax band bonding die is sprawled；
Excessive moisture is absorbed with filter paper, glass slide is put into 37 DEG C of incubator and bakes piece for 24 hours；
(9) dewaxing and rehydration:
Before dewaxing, glass slide is put into 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) operation 3 times in step 1) are repeated；
3) after dewaxing, 20s is kept in 100% ethyl alcohol；
4) 20s is kept in 95% ethyl alcohol；
5) 20s is kept in 70% ethyl alcohol；
6) 20s is kept in distilled water；
(10) dyeing and mounting:
Glass slide after taking dewaxing, rehydration is dyed using the fast green decoration method of sarranine-, and concrete operations are as follows:
1) 15min is kept in 1% sarranine aqueous solution；
2) 10s is kept in distilled water；
3) 10s is kept in 70% ethyl alcohol；
4) operation 3 times in step 3) are repeated；
5) 30s is kept in 0.5% 95% fast green ethanol solution；
6) 10s is kept in 95% ethyl alcohol；
7) operation 1 time in step 6) is repeated；
8) 10s is kept in dehydrated alcohol；
9) operation 1 time in step 8) is repeated；
10) after dyeing, with neutral gum mounting.
The micrograph in above-mentioned Picea meyeri (hardness of wood is I grade) stem tissue cross section being prepared is shown in Fig. 1.1 is in figure
Bast, 2 be forming layer, and 3 be xylem, and scale is 100 μm in figure.
Embodiment 2
The paraffin section method of trees stem tissue in the present embodiment, except acquisition 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 repaired in step (7) is put into 30 DEG C
Soften outside 2d in thermostat water bath, other operations are the same as embodiment 1.
The micrograph in above-mentioned Larix principis-rupprechtii (hardness of wood is III grade) stem tissue cross section being prepared is shown in Fig. 1.
1 is bast in figure, and 2 be forming layer, and 3 be xylem, and scale is 100 μm in figure.
Embodiment 3
The paraffin section method of trees stem tissue in the present embodiment, except acquisition cork oak (Quercus in step (1)
Variabilis Bl.) stem tissue (since cork oak bark is thicker, need to suitably strip outer bark) at the diameter of a cross-section of a tree trunk 1.3 meters above the ground, in step (7)
The embedded block after block will be repaired and be put into 30 DEG C of thermostat water baths and softened outside 7d, other operations are the same as embodiment 1.
The micrograph in above-mentioned cork oak (hardness of wood is IV~V grade) stem tissue cross section being prepared is shown in Fig. 1.
1 is bast in figure, and 2 be forming layer, and 3 be xylem, and scale is 100 μm in figure.
Illustrate: Wen Zhongyu ethyl alcohol relevant " % " all refers to volume fraction, 1% sarranine and 0.5% 95% fast green ethyl alcohol
" % " in solution all refers to mass fraction.
Claims (4)
1. a kind of paraffin section method of trees stem tissue, it is characterised in that: the following steps are included:
(1) tissue is fixed:
Trees stem tissue is dipped in fixer fixed；Stem tissue is derived from tree breast-height diameter in step (1), and tissue size is
1.5 ~ 2.5mm of diameter, 15 ~ 20mm of length；The stem tissue includes bast, forming layer and 3~5 years xylem regions；
(2) primary softening:
Tissue after fixation is dipped in 70% ethyl alcohol of volume ratio 1:1 and the bating liquor of glycerol and is softened；Soften in step (2)
Time is 1 ~ 4 week；
(3) tissue dewatering:
Concrete operations are as follows: 1) keep 1h in 35% ethyl alcohol；2) 1h is kept in 70% ethyl alcohol；3) it is kept in 90% ethyl alcohol
50min；4) it repeats to operate 1 time in step 3)；5) 50min is kept in 95% ethyl alcohol；6) 40min is kept in 100% ethyl alcohol；7)
It repeats to operate 1 time in step 6)；
(4) transparency of organization:
Concrete operations are as follows: 1) keep 40min in the mixed liquor of 100% ethyl alcohol of volume ratio 1:1 and dimethylbenzene；2) in dimethylbenzene
Middle holding 40min；3) it repeats to operate 1 time in step 2；
(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 30 DEG C of water half an hour to soften to one week；
(7) it is sliced, opens up piece, bonding die and roasting piece；
(8) dewaxing and rehydration:
After biopsy tissues dimethylbenzene dewaxing treatment, then the second alcohol and water rehydration for being successively 100%, 95%, 70% with concentration gradient；
The concrete operations of rehydration are as follows: 1) keep 20s in 100% ethyl alcohol；2) 20s is kept in 95% ethyl alcohol；3) it is kept in 70% ethyl alcohol
20s；4) 20s is kept in distilled water；
(9) dyeing and mounting: using the fast green decoration method dyeing of sarranine-, mounting；The concrete operations of dyeing are as follows: 1) at 1% kind
15 ~ 20min is kept in red aqueous solution；2) 10s is kept in distilled water；3) 10s is kept in 70% ethyl alcohol；4) step 3) is repeated
Middle operation 3 times；5) 30s is kept in 0.5% 95% fast green ethanol solution；6) 10s is kept in 95% ethyl alcohol；7) step is repeated
6) operation 1 time in；8) 10s is kept in dehydrated alcohol；9) it repeats to operate 1 time in step 8).
2. according to the method described in claim 1, it is characterized by: fixer by volume ratio is 9:0.5:0.5 in step (1)
70% ethyl alcohol, formaldehyde and acetic acid composition.
3. according to the method described in claim 1, it is characterized by: in step (5) waxdip concrete operations are as follows:
1) 20min is kept in atoleine；
2) 30min is kept in atoleine；
3) it repeats to operate 1 time in step 2.
4. according to the method described in claim 1, it is characterized by: the concrete operations to dewax in step (8) are as follows:
1) 5 ~ 10min is kept in dimethylbenzene；
2) it repeats to operate 3 times in step 1).
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| CN107167350B (en) * | 2017-05-16 | 2020-04-21 | 云南省农业科学院园艺作物研究所 | Preparation method of paraffin section of eggplant rhizome tissue |
| CN107702955B (en) * | 2017-08-24 | 2020-04-07 | 胡雨逸 | Preparation method of paraffin section |
| CN107843470B (en) * | 2017-10-13 | 2021-01-15 | 济南金域医学检验中心有限公司 | Method for making skin tissue slice |
| CN109115573B (en) * | 2018-08-27 | 2021-06-25 | 河南牧业经济学院 | Paraffin flaking method for xanthoceras sorbifolia cutting branches and callus thereof |
| CN109632425A (en) * | 2019-01-11 | 2019-04-16 | 北京林业大学 | A kind of paraffin section production method of Chinese herbaceous peony underground rhizome |
| CN110926851B (en) * | 2019-11-15 | 2022-03-25 | 华南农业大学 | Method for obtaining vascular cambium of woody plant and application thereof |
| CN110954384A (en) * | 2019-12-16 | 2020-04-03 | 贵州大学 | Preparation method of masson pine resin tract paraffin section |
| CN111238892B (en) * | 2020-01-19 | 2023-02-24 | 宁夏大学 | Permanent flaking method of lichen sporophore microstructure |
| CN111257089B (en) * | 2020-02-18 | 2020-10-27 | 广东省农业科学院环境园艺研究所 | Optimized manufacturing method of paraffin sections of different flower tissues of butterfly orchid |
| CN114088480A (en) * | 2020-08-25 | 2022-02-25 | 石河子大学 | Preparation method of paraffin sections of root and stem tissues of asafetida |
| CN112284791B (en) * | 2020-10-16 | 2023-02-28 | 扬州大学 | Method for separating xylem duct of grape fruit |
| CN114235536B (en) * | 2021-12-24 | 2024-03-19 | 贵州大学 | Slice preparation method for observing lignin deposition of stems of two-year-old masson pine |
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