CN107702959A - A kind of paraffin section method for vegetable material serial dehydration - Google Patents
A kind of paraffin section method for vegetable material serial dehydration Download PDFInfo
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- CN107702959A CN107702959A CN201711026813.8A CN201711026813A CN107702959A CN 107702959 A CN107702959 A CN 107702959A CN 201711026813 A CN201711026813 A CN 201711026813A CN 107702959 A CN107702959 A CN 107702959A
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- wax
- 5min
- ethanol
- dimethylbenzene
- paraffin
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Links
- 239000012188 paraffin wax Substances 0.000 title claims abstract description 53
- 230000018044 dehydration Effects 0.000 title claims abstract description 29
- 238000006297 dehydration reaction Methods 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 20
- 239000005418 vegetable material Substances 0.000 title claims abstract description 10
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims abstract description 113
- 239000000463 material Substances 0.000 claims abstract description 103
- 238000001035 drying Methods 0.000 claims abstract description 3
- 238000007789 sealing Methods 0.000 claims abstract description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 215
- 239000001993 wax Substances 0.000 claims description 83
- 235000019441 ethanol Nutrition 0.000 claims description 72
- 239000004575 stone Substances 0.000 claims description 37
- 239000000243 solution Substances 0.000 claims description 23
- 239000011259 mixed solution Substances 0.000 claims description 21
- 238000012545 processing Methods 0.000 claims description 21
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 12
- 238000004043 dyeing Methods 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 239000002023 wood Substances 0.000 claims description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims description 9
- 239000002245 particle Substances 0.000 claims description 9
- 239000008096 xylene Substances 0.000 claims description 7
- 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 6
- 235000011187 glycerol Nutrition 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 244000283070 Abies balsamea Species 0.000 claims description 5
- 235000007173 Abies balsamea Nutrition 0.000 claims description 5
- 239000004858 Canada balsam Substances 0.000 claims description 5
- 229960000583 acetic acid Drugs 0.000 claims description 5
- 239000011248 coating agent Substances 0.000 claims description 5
- 238000000576 coating method Methods 0.000 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 5
- 238000007711 solidification Methods 0.000 claims description 5
- 230000008023 solidification Effects 0.000 claims description 5
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 4
- 238000010257 thawing Methods 0.000 claims description 4
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 229920002866 paraformaldehyde Polymers 0.000 claims description 3
- 239000012362 glacial acetic acid Substances 0.000 claims description 2
- 238000005520 cutting process Methods 0.000 claims 1
- 238000000386 microscopy Methods 0.000 abstract description 6
- 230000008439 repair process Effects 0.000 abstract description 5
- 210000004027 cell Anatomy 0.000 abstract description 4
- 229930002875 chlorophyll Natural products 0.000 abstract description 3
- 235000019804 chlorophyll Nutrition 0.000 abstract description 3
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 abstract description 3
- 210000000170 cell membrane Anatomy 0.000 abstract description 2
- 210000003855 cell nucleus Anatomy 0.000 abstract description 2
- 241000196324 Embryophyta Species 0.000 description 22
- 239000000975 dye Substances 0.000 description 13
- 239000002131 composite material Substances 0.000 description 7
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 6
- 239000004411 aluminium Substances 0.000 description 5
- 229910052782 aluminium Inorganic materials 0.000 description 5
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000010586 diagram Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- 206010050808 Hyperchromasia Diseases 0.000 description 2
- 239000006059 cover glass Substances 0.000 description 2
- 239000006260 foam Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 1
- HDYRYUINDGQKMC-UHFFFAOYSA-M acetyloxyaluminum;dihydrate Chemical compound O.O.CC(=O)O[Al] HDYRYUINDGQKMC-UHFFFAOYSA-M 0.000 description 1
- 229940009827 aluminum acetate Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003610 charcoal Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005868 electrolysis reaction Methods 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000029052 metamorphosis Effects 0.000 description 1
- 229920001206 natural gum Polymers 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000007306 turnover Effects 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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- 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
- G01N2001/305—Fixative compositions
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Sampling And Sample Adjustment (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a kind of paraffin section method for vegetable material serial dehydration, including the collection and fixation of sample, the dehydration of sample, the transparent of sample, waxdip, embed, repair the steps such as block, fixation, section, exhibition piece, drying, dewaxing, sealing.The specimen material of the present invention is after dimethylbenzene is transparent, although the chlorophyll in plant can be degradable, but the cell membrane and nucleus of plant cell dye red by sarranine, and it is red that plant sample is presented, after dimethylbenzene is transparent, the color of paraffin of the color of sample with embedding easily is distinguished, embedding is easy in embedded samples into the sample direction of needs, and it can be clear from seeing position of the material in embedded block clearly during repairing block, make to repair block, section, open up piece and microscopic examination is very easy, it can avoid accidentally injuring material, the expansion degree of material is easily detected in microscopy, and then improve chipping qualities and conventional efficient.
Description
Technical field
The present invention relates to botany technical field, more particularly to a kind of paraffin section side for vegetable material serial dehydration
Method.
Background technology
Paraffin section is histology, the main experimental methods of Developmental Biology research, while is also that disease is observed in pathology
The important means of change is managed, outstanding contribution is made that for scientific research and clinical diagnosis.The technology application paraffin and the tissue of animals and plants
This general principle can be combined well, by collection of specimens, fixation, dehydration, transparent, waxdip, embedding, section, stand piece, patch
Transparent thin slice is made in a series of special methods such as piece, baking piece, dewaxing, dyeing, dehydration, transparent, in microscope or electronic display
Micro- Microscopic observation, it can faithfully reflect the structure and metamorphosis of body；The quantitatively size of institutional framework, number in determination sample
Amount and contained substance amount number；Meanwhile structure or contained substance are gone back under observable body mirror under different experimental conditions
Change, thus understand histiocytic activity, differentiation and intercellular correlation etc..The manufacturing process of paraffin section is not
Complexity, but it is many to influence the factor in terms of chipping qualities and efficiency, to make the section of high quality, each operation step
It is rapid all most important.
Processing procedure before paraffin section embedding is extremely important, flow before embedding include collection of specimens, fixation, dehydration,
Transparent, waxdip flow, process seem simple, but this determines the embedding of paraffin section later stage, repaiies block, section, stand piece, paster, exhibition piece
Efficiency, or even success or failure.
In practice after collection of specimens, ladder is carried out after the fixers such as FAA, Carnot's reagent, paraformaldehyde fix certain time
Spend dehydration of alcohol (30%, 50%, 70%, 85%, 90%, 100%, 100%), 1/2 alcohol and 1/2 dimethylbenzene it is excessive, pure two
Toluene is transparent twice, and for plant sample through fixation, gradient alcohol dehydration, the chlorophyll in plant can be degradable, and makes plant
Khaki or faint yellow is presented in sample, along with after dimethylbenzene is transparent, its sample especially flower, leaf and fresh and tender stem or root
Color of the color usually with the paraffin of embedding is difficult to differentiate between.In embedded samples, on the one hand because of color sample and paraffin color phase
Seemingly it is difficult to embedding into the sample direction of needs；On the other hand, even if the direction and sectional position and in the right direction of sample embedding,
Position of the material in embedded block is not often seen when repairing block and is difficult to repair block；Even if being smoothly cut into intact wax band in addition,
, also can be similar with paraffin because of material color when opening up piece, difficult microscopic, it is impossible to wax band is deployed well well, or exhibition piece
Excessively, sample exhibition is split, and then reduces chipping qualities or cause section destruction, reduces the efficiency of experiment.
At present in Technique of Paraffin Section, often through adding appropriate sarranine pair in 70% alcohol in dehydration
Material is dyed.However, added in 70% alcohol in dehydration after appropriate sarranine dyes to material, although after
The efficiency cut into slices during the embedding of phase, section and exhibition piece is improved, but kind of the one side plant sample through 70% alcohol
After red solution dehydrates, alcohol concentration can substantially reduce, can not again dehydration dye in use, therefore dissolved with sarranine alcohol most
It can still be thrown away eventually, the problems such as causing the waste of dyestuff, while cause the pollution of environment.On the other hand, 70% alcohol (contain
Turn over red) in dehydration often overlong time, cause plant sample hyperchromasia, and then some of later stage may be influenceed
Immunolocalization experiment effect.
The content of the invention
In view of this, it is an object of the invention to provide a kind of paraffin section method for vegetable material serial dehydration, make
Dyestuff during paraffin section material dehydration repeats utilization, dye loss is few, will not cause the pollution of environment, and monoblock is planted
Thing sample dye level is easily-controllable, and the immunolocalization experiment effect in later stage is good.
A kind of paraffin section method for vegetable material serial dehydration, including step in detail below：
1) collection and fixation of sample：Gather fresh material to immerse in fixer, vacuum suction 15~20min, Ran Houyu
24~36h is fixed in fixer, ensures the abundant soak-out material of fixer, fixer is killed cell rapidly, prevent aqtocytolysis,
Preserve complete eucaryotic cell structure；
2) dehydration of sample：By the material after fixation pass sequentially through mass fraction for 30%, 50%, 70%, 85%,
90%th, 100%, 100% graded ethanol solutions are dehydrated, and 0.2~2h is dehydrated respectively in the ethanol solution of each gradient；It is de-
The water time should regard the different and different of tissue types, tissue block size, thickness and fixative.
3) transparent and dyeing：By the material after dehydration prior to 1/2 absolute ethyl alcohol and 1/2 dimethylbenzene and 0.05wt%~
10~30min of transparent processing in 0.1wt% sarranine mixed solution, in pure xylene solution transparent processing twice, every time
10min is handled, to eliminate ethanol；
4) waxdip and embedding：30min in 1/2 dimethylbenzene and 1/2 paraffin mixed liquor is immersed the material into after transparent, is placed into molten
After point is 1~2h of waxdip in 52~56 DEG C of melted pure wax, the material after waxdip is put into 58 DEG C of insulating box and is incubated, is gone forward side by side
Row routine paraffin wax is embedded, and wax box is filled into melt paraffin, is moved into tweezers gripping material in box in paraffin, by being cut to obtain direction
Good material is put, and being blown on wax face makes the surface of wax congeal into thin layer, and then wax box is moved into cold water and is pressed into carton
Wax stone is obtained behind the bottom, completely solidification.
5) block and fixation are repaiied：The carton for including material and wax stone is taken out, the paper around coating wax stone is removed, exposes and include
There is a wax stone of material, according to material present position, wax stone cut and accomplishes low wide and up narrow trapezoidal, each fritter is included one
Individual material, and make Upper rectangular opposite side parallel, the wax of a few drop thawings is dripped on wood particle, and trapezoidal wax stone bottom is pasted on small wood
On block, make its firm pasting.
6) cut into slices, open up piece and drying：By trapezoidal wax stone in carrying out serial section, slice thickness 6 on rotary microtom
~10 μm, then serial section is put on slide, then the slide for being equipped with section is placed on 40~48 DEG C of exhibition piece platform,
After wax band is fully deployed, the unnecessary moisture that inclines inclines unnecessary moisture, room temperature dry or be placed in dried in 35 DEG C of baking ovens it is standby
With.
7) dewax, redye and sealing：Fully dry section is put into dimethylbenzene (twice) 20~30min of dewaxing treatment,
Through 1/2 dimethylbenzene and 1/2 absolute ethyl alcohol, absolute ethyl alcohol (twice), 95%, 85%, 70% (turning over red O dyeing containing 0.5-1%),
85%th, 95% (0.5-1%'s is fast green) ethanol, absolute ethyl alcohol (twice), 1/2 absolute ethyl alcohol and 1/2100% dimethylbenzene, diformazan
After benzene, dimethylbenzene, slide is pulled out, drip canada balsam in the middle part of material, covered, Permanent production is made.Its
In, natural gum can be diluted to suitable viscosity with dimethylbenzene, and its viscosity is to be easy to exclude bubble and in cover glass and section
Between can flow freely preferably, it is slightly thicker, can avoid dry after form the air gap under cover glass.
Further, fixer is one kind in Carnot's reagent, paraformaldehyde solution, FAA mixed solutions, and wherein FAA is mixed
Solution is formalin:Glacial acetic acid:70% ethanol presses 1:1:18 volume ratio is mixed to prepare.
Further, then 3~5 drop distilled water are added dropwise before use, prior to smearing glycerine albumen on slide in slide.
Using albumen glycerine as bonding die agent, to reach the purpose for reducing piece.
Further, the rehydration in step 7), the method redyed and be dehydrated are：Section after dewaxing is passed sequentially through 1/2 2
Toluene and 1/2 3~5min of absolute ethyl alcohol, 3~5min of absolute ethyl alcohol, 3~5min of absolute ethyl alcohol, 95% ethanol 3~5min, 85%
3~5min of ethanol, 70% ethanol and 0.5wt%~1wt% 0.2~2h of sarranine, 85% 3~5min of ethanol, 95% ethanol with
0.5wt%~1wt% fast green 3~10s, 3~5min of absolute ethyl alcohol, 3~5min of absolute ethyl alcohol, 1/2 dimethylbenzene and 1/2 are anhydrous
3~5min of ethanol, transparent processing is carried out into pure dimethylbenzene after degree.
In addition, waxdip is preferably foamed aluminium-paraffin composite with the paraffin used in embedding, and using foamed aluminium as skeleton, profit
Filling method is melted with paraffin vacuum paraffin is filled into the hole of foamed aluminium.In foamed aluminium-paraffin composite heat transfer process, stone
Wax increases with the good charcoal skeleton passage of heat contact area of heat conductivility, effectively reduces solid-liquid in paraffin refined wax diabatic process
Thermal resistance caused by boundary moves so that system heat transfer rate is significantly increased.Foamed aluminium-paraffin composite is in lower temperature
Lower fusing is more thorough, and temperature is more constant in molten foam aluminium-paraffin composite, foamed aluminium-paraffin composite wood during waxdip
Material can be penetrated into equably inside material structure, and is in close contact with material structure, prevent that tissue and foamed aluminium-paraffin are compound
Material departs from, and occurs bubble, the situation in crack in foamed aluminium-paraffin composite.In addition, steeped after the completion of in embedding
Foam aluminium-paraffin composite can fast cooling solidification, will not the material structure block caused by temperature is too high be hardened and become fragile and receive
Contracting, chipping qualities is high, and condensation is overrun and also will not cause wax stone rhegma because of internal-external temperature difference is excessive.
In dewaxing, fully dry section is first put into dimethylbenzene (twice) 20~30min of dewaxing treatment, then lead to successively
Cross 1/2 dimethylbenzene and 1/2 20~30min of acetic acid, pure 20~30min of acetic acid fully remove foamed aluminium, then carry out rehydration again, answer
Dye and dehydration process steps.Foamed aluminium and acetic acid reaction generation aluminum acetate, are recoverable aluminium by electrolysis, realize resource again
Utilize.
Beneficial effects of the present invention：
1. the specimen material of the present invention is after dimethylbenzene is transparent, although the chlorophyll in plant can be degradable, plant
The cell membrane and nucleus of thing cell dye red by sarranine, and it is red that plant sample is presented, after dimethylbenzene is transparent, sample
Color and the color of paraffin of embedding easily distinguish, embedding is easy in embedded samples into the sample direction of needs, and
It can be clear from seeing position of the material in embedded block clearly during repairing block, make to repair block, section and very easy, the energy of exhibition piece operation
Enough avoid accidentally injuring material, the expansion degree of material is easily detected in microscopy, and then improve chipping qualities and experiment effect
Rate；
The characteristics of 2. the specimen material of the present invention dissolves in ethanol using sarranine, and ethanol and can is dissolved in dimethylbenzene, general kind
It is red to add in 1/2 alcohol and 1/2 dimethylbenzene to realize the dyeing to material；Will not be molten because of sarranine of the plant sample through 70% alcohol
After fluid dewatering, alcohol concentration can substantially reduce, and finally can be still thrown away dissolved with the alcohol of sarranine and waste dyestuff and pollution environment
The problems such as, and 1/2 absolute ethyl alcohol and 1/2 dimethylbenzene are transparent as pure xylene solution with the processing of 0.1wt%~0.2wt% sarranine
The transition step of processing, mixed liquor, which may be reused, is many times improved, the service efficiency of dyestuff；In addition, material in
1/2 absolute ethyl alcohol and 1/2 dimethylbenzene and transparent processing time in 0.05wt%~0.1wt% sarranine mixed solutions are short, Bu Huizao
Into plant sample hyperchromasia.
Brief description of the drawings
Fig. 1 is after being dyed by 1/2 absolute ethyl alcohol and 1/2 dimethylbenzene with 0.05wt%~0.1wt% sarranine mixed solution
Materials behavior figure；
Fig. 2 be no dyeing material after FFPE after materials behavior figure；
Fig. 3 is the material dyed by 70% alcohol and 0.05wt%~0.1wt% sarranine mixed solution through FFPE
Sample drawing afterwards；
Fig. 4 is to be dyed by 1/2 absolute ethyl alcohol and 1/2 dimethylbenzene with 0.05wt%~0.1wt% sarranine mixed solutions
Sample drawing of the material after FFPE；
Wherein,
Figure 1A is to be dyed by 1/2 absolute ethyl alcohol and 1/2 dimethylbenzene with 0.05wt%~0.1wt% sarranine mixed solutions
Schematic diagram of the material after FFPE；
Figure 1B is to be dyed by 1/2 absolute ethyl alcohol and 1/2 dimethylbenzene with 0.05wt%~0.1wt% sarranine mixed solutions
Wax band figure after material is sliced；
Fig. 2A is schematic diagram of the material of no dyeing after FFPE；
Fig. 2 B be the material of no dyeing it is sliced after wax band figure；
Fig. 3 A are the materials dyed by 70% alcohol and 0.05wt%~0.1wt% sarranine mixed solution through FFPE
Schematic diagram afterwards；
Fig. 3 B be the material dyed by 70% alcohol and 0.05wt%~0.1wt% sarranine mixed solution it is sliced after
Wax band microscopy figure；
Fig. 4 A are to be dyed by 1/2 absolute ethyl alcohol and 1/2 dimethylbenzene with 0.05wt%~0.1wt% sarranine mixed solutions
Schematic diagram of the material after FFPE；
Fig. 4 B are to be dyed by 1/2 absolute ethyl alcohol and 1/2 dimethylbenzene with 0.05wt%~0.1wt% sarranine mixed solutions
Wax band microscopy figure after material is sliced.
Embodiment
Below with reference to embodiment, the present invention is described in detail：
Embodiment one
Gather fresh material to immerse in Carnot's reagent, 15~20min of vacuum suction, then fix 24 in Carnot's reagent~
36h；Material after fixation is passed sequentially through into the ladder that mass fraction is 30%, 50%, 70%, 85%, 90%, 100%, 100%
Degree ethanol solution is dehydrated, and 0.2~2h is dehydrated respectively in the ethanol solution of each gradient；By the material after dehydration prior to 1/2
Absolute ethyl alcohol and 1/2 dimethylbenzene and 10~30min of transparent processing in 0.05wt%~0.1wt% sarranine mixed solution, then at
In pure xylene solution transparent processing twice, 10min per treatment；1/2 dimethylbenzene is immersed the material into after transparent to mix with 1/2 paraffin
30min in liquid is closed, after placing into fusing point as 1~2h of waxdip in 52~56 DEG C of melted pure wax, the material after waxdip is put into 58
DEG C insulating box in be incubated, wax box is then filled into melt paraffin, is moved into box in paraffin, is well placed with tweezers gripping material,
And being blown on wax face makes the surface of wax congeal into thin layer, then wax box is moved into cold water and carton is pressed into water-bed, solidification completely
Obtain wax stone afterwards；The carton for including material and wax stone is taken out, the paper around coating wax stone is removed, exposes the wax for including material
Block, according to material present position, wax stone is cut and accomplishes low wide and up narrow trapezoidal, each fritter is included a material, and
Make Upper rectangular opposite side parallel, drip the wax of a few drop thawings on wood particle, trapezoidal wax stone bottom is pasted on wood particle, makes it
Firm pasting；By trapezoidal wax stone in carrying out serial section on rotatable slicer, slice thickness is 6~10 μm, then will continuously be cut
Piece, which is put into, is coated with glycerine albumen, and is added dropwise on 3~5 drop distilled water slides, then the slide for being equipped with section is placed on 40~
On 48 DEG C of exhibition piece platform, after wax band is fully deployed, incline unnecessary moisture, room temperature dry or be placed in dried in 35 DEG C of baking ovens it is standby
With；Fully dry section is put into dimethylbenzene 20~30min of dewaxing treatment, the section after dewaxing is then passed sequentially through 1/2
Dimethylbenzene and 1/2 3~5min of absolute ethyl alcohol, 3~5min of absolute ethyl alcohol, 3~5min of absolute ethyl alcohol, 95% 3~5min of ethanol,
85% 3~5min of ethanol, 70% ethanol and 0.5wt%~1wt% 0.2~2h of sarranine, 85% 3~5min of ethanol, 95% second
Fast green 3~10s, 3~5min of absolute ethyl alcohol, 3~5min of absolute ethyl alcohol, 1/2 dimethylbenzene and 1/2 of alcohol and 0.5wt%~1wt%
3~5min of absolute ethyl alcohol, after degree transparent processing 2 times into pure dimethylbenzene, 3~5min, then pulls slide, Yu Cai out every time
Canada balsam is dripped at material middle part, covered, Permanent production is made.
Passed through by 1/2 absolute ethyl alcohol and 1/2 dimethylbenzene with the material that 0.05wt%~0.1wt% sarranine mixed solution dyes
For state after FFPE as shown in Figure 1A and Fig. 4 A, plant sample color is light, but plant sample and paraffin can apparent areas
Do not open.The material embedded after dyeing, it is sliced after wax band as shown in Figure 1B, plant sample and paraffin significant difference can be opened.
The result of plant section microscopy under microscope made of material in wherein Fig. 4 A after FFPE as shown in Figure 4 B, plant
Sample wax band color is light, but still is easily discriminated the details of section, easily detects the expansion degree of material.In addition, 1/2 nothing
Water-ethanol and 1/2 dimethylbenzene are handled with 0.1wt%~0.2wt% sarranine to be walked as the transition of pure xylene solution transparent processing
Suddenly, mixed liquor, which may be reused, is many times improved, the service efficiency of dyestuff.
Embodiment two
Gather fresh material to immerse in Carnot's reagent, 15~20min of vacuum suction, then fix 24 in Carnot's reagent~
36h；Material after fixation is passed sequentially through into mass fraction as 30%, 50%, 70% ethanol and 0.05wt%~0.1wt% kind
Red, 85%, 90%, 100%, 100% graded ethanol solutions are dehydrated, and are dehydrated respectively in the ethanol solution of each gradient
0.2~2h；By the material after dehydration prior to 10~30min of transparent processing in 1/2 absolute ethyl alcohol and 1/2 dimethylbenzene mixed solution,
In pure xylene solution transparent processing twice, 10min per treatment；1/2 dimethylbenzene and 1/2 stone are immersed the material into after transparent
30min in wax mixed liquor, after placing into fusing point as 1~2h of waxdip in 52~56 DEG C of melted pure wax, the material after waxdip is put
Enter and be incubated in 58 DEG C of insulating box, wax box is then filled into melt paraffin, with paraffin in tweezers gripping material immigration box, put
It is good, and being blown on wax face makes the surface of wax congeal into thin layer, then wax box is moved into cold water and carton is pressed into the bottom, it is completely solidifying
Gu wax stone is obtained after；The carton for including material and wax stone is taken out, the paper around coating wax stone is removed, exposes and include material
Wax stone, according to material present position, wax stone is cut and accomplishes low wide and up narrow trapezoidal, each fritter is included a material,
And make Upper rectangular opposite side parallel, the wax of a few drop thawings is dripped on wood particle, and trapezoidal wax stone bottom is pasted on wood particle, made
Its firm pasting；By trapezoidal wax stone in carrying out serial section on rotatable slicer, slice thickness is 6~10 μm, then will be continuous
Section, which is put into, is coated with glycerine albumen, and is added dropwise on 3~5 drop distilled water slides, then the slide for being equipped with section is placed on into 40
On~48 DEG C of exhibition piece platform, after wax band is fully deployed, incline unnecessary moisture, and room temperature is dried or is placed in dries in 35 DEG C of baking ovens
It is standby；Fully dry section is put into dimethylbenzene 20~30min of dewaxing treatment, the section after dewaxing is then passed sequentially through 1/
2 dimethylbenzene and 1/2 3~5min of absolute ethyl alcohol, 3~5min of absolute ethyl alcohol, 3~5min of absolute ethyl alcohol, 95% 3~5min of ethanol,
85% 3~5min of ethanol, 70% ethanol and 0.5wt%~1wt% 0.2~2h of sarranine, 85% 3~5min of ethanol, 95% second
Fast green 3~10s, 3~5min of absolute ethyl alcohol, 3~5min of absolute ethyl alcohol, 1/2 dimethylbenzene and 1/2 of alcohol and 0.5wt%~1wt%
3~5min of absolute ethyl alcohol, after degree transparent processing 2 times into pure dimethylbenzene, 3~5min, then pulls slide, Yu Cai out every time
Canada balsam is dripped at material middle part, covered, Permanent production is made.
The material dyed by 70% ethanol and 0.05wt%~0.1wt% sarranine mixed solution is after FFPE
As shown in Figure 3A, because dewatering time is longer, plant sample dyeing is deep after embedding for state.Material system in Fig. 3 A after FFPE
Into plant section microscopy under microscope result as shown in Figure 3 B, because dewatering time is longer, after embedded section, plant sample
Product color, the details of section, which is distinguished, to be not easy, it is difficult to detects the expansion degree of material.In addition, plant sample is through 70% alcohol
After sarranine solution dehydrates, alcohol concentration can substantially reduce, and finally can be still thrown away dissolved with the alcohol of sarranine and waste dyestuff and dirt
The problems such as contaminating environment.
Embodiment three
Gather fresh material to immerse in Carnot's reagent, 15~20min of vacuum suction, then fix 24 in Carnot's reagent~
36h；Material after fixation is passed sequentially through into the ladder that mass fraction is 30%, 50%, 70%, 85%, 90%, 100%, 100%
Degree ethanol solution is dehydrated, and 0.2~2h is dehydrated respectively in the ethanol solution of each gradient；By the material after dehydration prior to 1/2
10~30min of transparent processing in absolute ethyl alcohol and 1/2 dimethylbenzene mixed solution, the transparent processing two in pure xylene solution
It is secondary, 10min per treatment；30min in 1/2 dimethylbenzene and 1/2 paraffin mixed liquor is immersed the material into after transparent, placing into fusing point is
In 52~56 DEG C of melted pure wax after 1~2h of waxdip, the material after waxdip is put into 58 DEG C of insulating box and is incubated, then by wax
Box fills melt paraffin, with paraffin in tweezers gripping material immigration box, is well placed, and being blown on wax face coagulates the surface of wax
Straticulation, then wax box is moved into cold water and carton is pressed into the bottom, obtain wax stone after solidification completely；Taking-up include material and
The carton of wax stone, the paper around coating wax stone is removed, exposes the wax stone for including material, according to material present position, by wax stone
Cut and accomplish low wide and up narrow trapezoidal, each fritter is included a material, and make Upper rectangular opposite side parallel, drip several drops and melt
Trapezoidal wax stone bottom is pasted on wood particle on wood particle, makes its firm pasting by the wax of change；Trapezoidal wax stone is cut in rotatable
Serial section is carried out on piece machine, slice thickness is 6~10 μm, is then put into serial section and is coated with glycerine albumen, and is added dropwise 3
~5 are dripped on distilled water slides, then the slide for being equipped with section is placed on 40~48 DEG C of exhibition piece platform, treat that wax band is fully deployed
Afterwards, incline unnecessary moisture, and room temperature is dried or is placed in be dried for standby in 35 DEG C of baking ovens；Fully dry section is put into dimethylbenzene
20~30min of dewaxing treatment, then by the section after dewaxing pass sequentially through 100% 3~5min of ethanol, 95% 3~5min of ethanol,
83% 3~5min of ethanol, 70% 3~5min of ethanol, 50% 3~5min of ethanol, 35% 3~5min of ethanol, 0.5wt%~
1wt% 1~12h of sarranine, 35% 3~5min of ethanol, 50% 3~5min of ethanol, 70% 3~5min of ethanol, 83% ethanol 3~
5min, 0.5wt%~1wt% fast green 3~10s, 95% 3~5min of ethanol, 100% 3~5min of ethanol, 1/2 dimethylbenzene with
1/2 3~5min of absolute ethyl alcohol, after degree transparent processing 2 times into pure dimethylbenzene, 3~5min, then pulls slide out every time,
Canada balsam is dripped in the middle part of material, covered, Permanent production is made.
State of the material of no dyeing after FFPE as shown in Figure 2 A, plant sample and paraffin face in embedded block
Color is difficult difference, it is difficult to is embedded into the sample direction of needs.The material embedded after dyeing, it is sliced after wax band such as Fig. 2 B institutes
Show, plant sample and paraffin color are difficult to distinguish, and position of the material in embedded block is not seen during repairing block and is difficult to repair block.
The above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although with reference to preferred embodiment to this hair
It is bright to be described in detail, it will be understood by those within the art that, technical scheme can be modified
Or equivalent substitution, without departing from the objective and scope of technical solution of the present invention, it all should cover the claim in the present invention
Among scope.The present invention be not described in detail technology, shape, construction part be known technology.
Claims (4)
1. a kind of paraffin section method for vegetable material serial dehydration, it is characterised in that including step in detail below：
1) collection and fixation of sample：Gather fresh material to immerse in fixer, 15~20min of vacuum suction, then in fixation
24~36h is fixed in liquid；
2) dehydration of sample：By the material after fixation pass sequentially through mass fraction for 30%, 50%, 70%, 85%, 90%,
100%th, 100% graded ethanol solutions are dehydrated, and 0.2~2h is dehydrated respectively in the ethanol solution of each gradient；
3) transparent and dyeing：By the material after dehydration prior to 1/2 absolute ethyl alcohol and 1/2 dimethylbenzene and 0.05wt%~0.1wt%
Sarranine mixed solution in 10~30min of transparent processing, transparent processing is twice, per treatment in pure xylene solution
10min；
4) waxdip and embedding：30min in 1/2 dimethylbenzene and 1/2 paraffin mixed liquor is immersed the material into after transparent, placing into fusing point is
In 52~56 DEG C of melted pure wax after 1~2h of waxdip, the material after waxdip is put into 58 DEG C of insulating box and is incubated, then by wax
Box fills melt paraffin, with paraffin in tweezers gripping material immigration box, is well placed, and being blown on wax face coagulates the surface of wax
Straticulation, then wax box is moved into cold water and carton is pressed into the bottom, obtain wax stone after solidification completely；
5) block and fixation are repaiied：The carton for including material and wax stone is taken out, the paper around coating wax stone is removed, exposes and include material
The wax stone of material, according to material present position, wax stone is cut and accomplishes low wide and up narrow trapezoidal, each fritter is included a material
Material, and make Upper rectangular opposite side parallel, the wax of a few drop thawings is dripped on wood particle, and trapezoidal wax stone bottom is pasted on wood particle
On, make its firm pasting；
6) cut into slices, open up piece and drying：By trapezoidal wax stone in carrying out serial section on rotatable slicer, slice thickness is 6~10 μm,
Then serial section is put on slide, then the slide for being equipped with section is placed on 40~48 DEG C of exhibition piece platform, treat wax band
After being fully deployed, incline unnecessary moisture, and room temperature is dried or is placed in be dried for standby in 35 DEG C of baking ovens；
7) dewax, redye and sealing：Fully dry section is put into dimethylbenzene 20~30min of dewaxing treatment, cutting after dewaxing
Piece carries out rehydration with graded ethanol, redyes and be dehydrated, after degree transparent processing 2 times into pure dimethylbenzene, 3~5min every time, so
After pull slide out, drip canada balsam in the middle part of the material, covered, Permanent production be made.
A kind of 2. paraffin section method for vegetable material serial dehydration according to claim 1, it is characterised in that institute
It is one kind in Carnot's reagent, paraformaldehyde solution, FAA mixed solutions to state fixer in step 1), and wherein FAA mixed solutions are
Formalin:Glacial acetic acid:70% ethanol presses 1:1:18 volume ratio is mixed to prepare.
A kind of 3. paraffin section method for vegetable material serial dehydration according to claim 1, it is characterised in that institute
The slide in step 6) is stated before use, prior to smearing glycerine albumen on slide, 3~5 drop distilled water are then added dropwise.
A kind of 4. paraffin section method for vegetable material serial dehydration according to claim 1, it is characterised in that institute
The method for state the rehydration in step 7), redying and being dehydrated is：Section after dewaxing is passed sequentially through into 1/2 dimethylbenzene and 1/2 anhydrous
3~5min of ethanol, 3~5min of absolute ethyl alcohol, 3~5min of absolute ethyl alcohol, 95% ethanol 3~5min, 85% 3~5min of ethanol,
70% ethanol and 0.5wt%~1wt% 0.2~2h of sarranine, 85% 3~5min of ethanol, 95% ethanol and 0.5wt%~
1wt% fast green 3~10s, 3~5min of absolute ethyl alcohol, 3~5min of absolute ethyl alcohol, 1/2 dimethylbenzene and 1/2 absolute ethyl alcohol 3~
5min, transparent processing is carried out into pure dimethylbenzene after degree.
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| CN108871842A (en) * | 2018-04-19 | 2018-11-23 | 北京林业大学 | A kind of free-hand section method and its application of Chinese herbaceous peony rhizome tissue |
| CN109374378A (en) * | 2018-11-22 | 2019-02-22 | 黑龙江大学 | A kind of method of beet tops paraffin embedding film-making |
| CN109991063A (en) * | 2019-04-25 | 2019-07-09 | 广西壮族自治区药用植物园 | The paraffin section method of Paris polyphylla root tuber |
| CN110951835A (en) * | 2019-12-30 | 2020-04-03 | 北京市园林科学研究院 | RNA in-situ hybridization method for herbaceous flowers |
| CN112798322A (en) * | 2020-12-23 | 2021-05-14 | 广东东阳光药业有限公司 | 3D cell microsphere paraffin section method |
| CN112945657A (en) * | 2021-01-22 | 2021-06-11 | 中国科学院东北地理与农业生态研究所 | Pretreatment device and method for preparing plant material paraffin section |
| CN113008648A (en) * | 2021-03-01 | 2021-06-22 | 北京林业大学 | Fluorescence microscopy method for observing behavior of peony pollen tube by combining improved paraffin section with aniline blue dyeing method |
| CN113039955A (en) * | 2021-01-22 | 2021-06-29 | 韶关学院 | Method for inhibiting loquat flowering |
| CN113358391A (en) * | 2021-06-02 | 2021-09-07 | 中国热带农业科学院热带生物技术研究所 | Method for slicing plant leaf paraffin |
| CN114923755A (en) * | 2022-05-27 | 2022-08-19 | 河北农业大学 | Method for separating Chinese cabbage histiocyte based on laser microdissection technology |
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| CN108871842A (en) * | 2018-04-19 | 2018-11-23 | 北京林业大学 | A kind of free-hand section method and its application of Chinese herbaceous peony rhizome tissue |
| CN109374378A (en) * | 2018-11-22 | 2019-02-22 | 黑龙江大学 | A kind of method of beet tops paraffin embedding film-making |
| CN109991063A (en) * | 2019-04-25 | 2019-07-09 | 广西壮族自治区药用植物园 | The paraffin section method of Paris polyphylla root tuber |
| CN110951835A (en) * | 2019-12-30 | 2020-04-03 | 北京市园林科学研究院 | RNA in-situ hybridization method for herbaceous flowers |
| CN112798322A (en) * | 2020-12-23 | 2021-05-14 | 广东东阳光药业有限公司 | 3D cell microsphere paraffin section method |
| CN112945657A (en) * | 2021-01-22 | 2021-06-11 | 中国科学院东北地理与农业生态研究所 | Pretreatment device and method for preparing plant material paraffin section |
| CN113039955A (en) * | 2021-01-22 | 2021-06-29 | 韶关学院 | Method for inhibiting loquat flowering |
| CN112945657B (en) * | 2021-01-22 | 2023-08-25 | 中国科学院东北地理与农业生态研究所 | Pretreatment device and method for preparing plant material paraffin section |
| CN113008648A (en) * | 2021-03-01 | 2021-06-22 | 北京林业大学 | Fluorescence microscopy method for observing behavior of peony pollen tube by combining improved paraffin section with aniline blue dyeing method |
| CN113358391A (en) * | 2021-06-02 | 2021-09-07 | 中国热带农业科学院热带生物技术研究所 | Method for slicing plant leaf paraffin |
| CN114923755A (en) * | 2022-05-27 | 2022-08-19 | 河北农业大学 | Method for separating Chinese cabbage histiocyte based on laser microdissection technology |
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