CN107389412A - A kind of paraffin section preparation method of Shrimp waste watery tissue - Google Patents
A kind of paraffin section preparation method of Shrimp waste watery tissue Download PDFInfo
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- CN107389412A CN107389412A CN201710768140.7A CN201710768140A CN107389412A CN 107389412 A CN107389412 A CN 107389412A CN 201710768140 A CN201710768140 A CN 201710768140A CN 107389412 A CN107389412 A CN 107389412A
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- 239000002699 waste material Substances 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
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- 238000000034 method Methods 0.000 claims abstract description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 232
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 54
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 238000001816 cooling Methods 0.000 claims description 18
- 238000012545 processing Methods 0.000 claims description 16
- 239000001993 wax Substances 0.000 claims description 16
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 14
- 229920002866 paraformaldehyde Polymers 0.000 claims description 14
- 239000000834 fixative Substances 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 11
- 208000005156 Dehydration Diseases 0.000 claims description 8
- 230000018044 dehydration Effects 0.000 claims description 8
- 238000006297 dehydration reaction Methods 0.000 claims description 8
- 239000012153 distilled water Substances 0.000 claims description 7
- 238000004043 dyeing Methods 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000002474 experimental method Methods 0.000 claims description 6
- 239000002184 metal Substances 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 238000011049 filling Methods 0.000 claims description 4
- 239000007836 KH2PO4 Substances 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 3
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 claims description 3
- 230000005484 gravity Effects 0.000 claims description 3
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
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- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Natural products C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 claims 1
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- 235000019441 ethanol Nutrition 0.000 description 58
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- 210000000514 hepatopancreas Anatomy 0.000 description 9
- 210000004347 intestinal mucosa Anatomy 0.000 description 8
- 238000005516 engineering process Methods 0.000 description 6
- 241000058338 Macrobrachium nipponense Species 0.000 description 5
- 230000008520 organization Effects 0.000 description 5
- 241000723353 Chrysanthemum Species 0.000 description 4
- 235000007516 Chrysanthemum Nutrition 0.000 description 4
- 210000004907 gland Anatomy 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- 210000003360 nephrocyte Anatomy 0.000 description 4
- 210000005239 tubule Anatomy 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
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- HLUCICHZHWJHLL-UHFFFAOYSA-N hematein Chemical compound C12=CC=C(O)C(O)=C2OCC2(O)C1=C1C=C(O)C(=O)C=C1C2 HLUCICHZHWJHLL-UHFFFAOYSA-N 0.000 description 2
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- 230000007812 deficiency Effects 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
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- 230000002440 hepatic effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 229920001206 natural gum Polymers 0.000 description 1
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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
- 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
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
-
- 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
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
- G01N2001/2873—Cutting or cleaving
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- General Physics & Mathematics (AREA)
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Abstract
The present invention relates to a kind of paraffin section preparation method of Shrimp waste watery tissue；Conventional paraffin tissue sections method index for the fixation of tissue morphology difficulty, soft texture, watery the inorganizable discrimination of Shrimp waste histoorgan, easily cause historrhexis, overlapping, warpage, have a strong impact on the microexamination effect of tissue, it is difficult to obtain preferable clearly institutional framework.The present invention and then shape tissue, extension tissue by avoiding fold overlapping the negative pressure of sample, fixation and embedding treatment and error being present because of the elasticity of watery tissue.The present invention is easy to operate, feasibility is strong, the cycle is short, for the Shrimp waste histoorgan of the fixation of tissue morphology difficulty, soft texture, watery there is good tissue area to index, the paraffin section observation of complete tissue morphology can be realized, whole requirements that researcher is observed biopsy tissues can be met.
Description
Technical field
The present invention relates to a kind of paraffin section preparation method of Shrimp waste watery tissue, belong to microscopic tissue sections technology
Field.
Background technology：
Shrimp waste tissue paraffin section de technology is to study a kind of important technology of animal tissue's form and Pathologic changes.By
It is aquatic animal in Shrimp waste, the water content of its various histoorgan is higher, especially passes through frequently as pathological research foundation
Gill tissue and hepatopancrease (mid intestinal gland) tissue.In recent years, Shrimp waste both the above is organized research process materials amount it is big,
Reagent dosage is more, and for economic consideration, conventional art is still used as and widely uses technology.Technology generally comprise materials and it is fixed,
Flowing water flushing, tissue dewatering, transparency of organization, ooze the steps such as wax embedding, section exhibition piece, dewaxing and HE dyeing, light microscopic microexamination.
And conventional method index for the fixation of tissue morphology difficulty, soft texture, watery the inorganizable area of Shrimp waste histoorgan
Indexing, easily causes historrhexis, overlapping, warpage, has a strong impact on the microexamination effect of tissue, it is difficult to obtains preferable clearly group
Knit structure.It is contemplated that making up existing method is handling the technical deficiency of the watery animals such as Shrimp waste and its tissue, to obtain
Obtain preferable histoorgan paraffin section image.
The content of the invention：
In order to solve the above problems, the invention provides a kind of paraffin section preparation method of Shrimp waste watery tissue.
A kind of paraffin section preparation method of Shrimp waste tissue, step are as follows：
1st, sample tissue is put in tissue fixative solution, negative pressure is fixed immediately, and negative pressure to sample tissue block expands full, no
Bubble is produced again；It is added to immediately again in the centrifuge tube of the appropriate volume for the tissue fixative solution for filling same concentrations, in centrifuge tube
The ratio between amount and sample tissue volume of tissue fixative solution are 20：1；Tissue block is avoided to be affixed on chamber wall, room temperature (20-25 DEG C)
Constant temperature fixes 4-6h；The pressure value of the negative pressure is -1.25kpa；Negative-pressure container total measurement (volume) (V1)：Tissue fixative solution is total in container
Volume (V2):Sample tissue volume (V3)=6:3:1；
The tissue fixative solution is the paraformaldehyde solution of Solute mass fraction 2.5%；Paraformaldehyde solution is specifically prepared
Method is：Paraformaldehyde 50g, 1 × PBS cushioning liquid 900ml is taken, adds water to 1000ml, water-bath 24h at 60 DEG C after mixing, if
Also undissolved paraformaldehyde, the NaOH solution of the equivalent (1mol/L) of 1-2 drops 1 is added dropwise, regulation PH is 6.8-7.2, is protected at 4 DEG C
Deposit；
The component of 1 × PBS cushioning liquid (0.01M) is：Na2HPO48mM, NaCl 136mM, KH2PO42mM,
KCl 2.6mM, PH=7.2-7.4；
The NaOH solution of 1 equivalent (1mol/L) is：NaOH solid powder 10g are weighed, are configured with 250mL volumetric flask
Into 250mL, 1mol/L NaOH solution.
2nd, sample tissue of the step 1) after fixed is put into embedded box, with the flowing uninterrupted flow wash embedded box of clear water
24h；Remove the fixer remained in sample tissue；Wrapped after flushing with lens wiping paper.
3rd, liquidate and wash complete sample tissue under normal temperature (25 DEG C or so), successively with gradient concentration be 30%, 40%,
50%th, 60%, 70%, 80%, 90%, 100% ethanol solution dewater treatment；Concentration is 30% ethanol solution dewater treatment
Time is respectively 4h-5h；The ethanol solution dehydration treatment time that concentration is 40%, 50%, 60%, 70% is respectively 40min-
1h, the ethanol dehydration processing time of concentration 80%, 90%, 100% is respectively 15min-35min；When ethanol solution concentration≤
When 70%, the volume ratio of the ethanol solution and sample tissue is 50:1；When ethanol solution concentration >=70%, the ethanol is molten
The volume ratio of liquid and sample tissue is 35:1；By the embedded box equipped with sample tissue from ethanol solution after the completion of dewater treatment
Middle taking-up.
4th, the embedded box taken out in step 3) is put into clarifier and carries out transparent processing.
5th, transparent good sample tissue is put into wax pan, wax 1h is oozed at 60 DEG C.Ooze with metal embedding device to enter after the completion of wax
Row embedding, embedding device is removed after complete cooled and solidified, preserved at next step experiment, or 4 DEG C.
6th, tighten and fix slicer head, consolidate cutter frame structure, adjustment slice thickness is 5 μm.Cut into slices first
Preliminary experiment is repaired, until after cutting out complete maximum tissue section, then carry out formal cutting.To cut into slices as far as possible it is smooth spread out, gently
Drag the thermostatted water surface layer for making its individual layer be laid on exhibition piece machine.Naturally dry 12h, 100 DEG C of oven for baking 2h.
The preliminary experiment finishing is carried out in three steps：First, section, ensures that the section that each knife is cut out all is 5 μm uniformly thick
The monolithic histotomy of degree, avoids the occurrence of uneven thickness；Second, cutting size, ensure the shape of cross section for the section that each knife is cut out
It is similar with area；Third, cut microstructure, by the way that section is constantly observed under the microscope, ensure that the section that cuts out is microcosmic
Tissue should meet crosscutting, rip cutting requirement, while high-visible.
7th, the section after drying is subjected to dewaxing treatment with dimethylbenzene II solution, after the completion of dewaxing with 100%, 95%,
90%th, 80%, 70%, 60% concentration gradient ethanol solution and Yihong-haematine dye liquor carry out dyeing processing；Finally, in
Property natural gum will dye the section completed and carry out mounting processing, and drying process is made into the section sealed with insulating box.
Preferably, tissue dewatering step is in the step 3)：
1) 25 DEG C, 4h is persistently handled in the ethanol of 30% concentration；
2) 25 DEG C, 40min is persistently handled in the ethanol of 40% concentration；
3) 25 DEG C, 40min is persistently handled in the ethanol of 50% concentration；
4) 25 DEG C, 40min is persistently handled in the ethanol of 60% concentration；
5) 25 DEG C, 40min is persistently handled in the ethanol of 70% concentration；
6) 25 DEG C, 40min is persistently handled in the ethanol of 80% concentration；
7) 25 DEG C, 40min is persistently handled in the ethanol of 90% concentration；
8) 25 DEG C, 40min is persistently handled in the ethanol of 95% concentration；
9) 25 DEG C, 15min is persistently handled in the ethanol of 100% concentration；
10) 25 DEG C, 15min is persistently handled in the ethanol of 100% concentration.
In the step 4) clarifier be Solute mass fraction be 50% the solution of dimethylbenzene I, Solute mass fraction be
95% solution of dimethylbenzene II.The solution component of dimethylbenzene I is 100% ethanol and 100% dimethylbenzene with volume ratio 1:1 is mixed
Close；The solution component of dimethylbenzene II is 100% ethanol and 100% dimethylbenzene with volume ratio 1:19 mixing.
Transparency of organization processing step is in the step 4)：
1) get rid of net remaining embedded box ethanol solution, with after hanging Action of Gravity Field 30s without ethanol solution drop drip for
Standard；
2) embedded box is first put into the solution of dimethylbenzene I and 30min in the solution of dimethylbenzene II is placed into after 40min.
Hydrodewaxing step is in the step 7)：Dry section is taken, it is 95% to be put in and fill Solute mass fraction
Dewax 10min in the solution of dimethylbenzene II.
It is the step of dyeing processing in the step 7)：
1) 25 DEG C, 100% ethanol solution persistently handles 10min；
2) 25 DEG C, 95% ethanol solution persistently handles 10min；
3) 25 DEG C, 90% ethanol solution persistently handles 10min；
4) 25 DEG C, 80% ethanol solution persistently handles 10min；
5) 25 DEG C, 70% ethanol solution persistently handles 10min；
6) 25 DEG C, 60% ethanol solution persistently handles 10min；
7) 25 DEG C, distilled water persistently handles 5min, 4 DEG C of cooling 3min；
8) 25 DEG C, haematine dye liquor persistently handles 10min, 4 DEG C of cooling 3min；
9) 25 DEG C, distilled water is gently stained with 1-2 times, 4 DEG C of cooling 1-2min；
10) 25 DEG C, color separation liquid persistently handles 5s, 4 DEG C of cooling 10s；11) 25 DEG C, deionized water is gently stained with 1-2 times, 4 DEG C of coolings
3min；The color separation liquid component is：The alcohol of Solute mass fraction 70% and 0.5% hydrochloric acid are with volume ratio 100:1 mixing；
12) 25 DEG C, eosin stain persistently handles 1min, 4 DEG C of cooling 10s；
13) 25 DEG C, 90% ethanol solution persistently handles 2min；
14) 25 DEG C, 95% ethanol solution persistently handles 2min；
15) 25 DEG C, 100% ethanol solution persistently handles 10min；
16) 25 DEG C, the solution of dimethylbenzene I that Solute mass fraction is 50% persistently handles 10min；
17) 25 DEG C, the solution of dimethylbenzene II that Solute mass fraction is 95% persistently handles 10min.
Beneficial effect of the present invention：
At present, conventional paraffin tissue sections method index for the fixation of tissue morphology difficulty, soft texture, watery shrimp
The inorganizable discrimination of crab class loading organ, historrhexis, overlapping, warpage are easily caused, have a strong impact on the microexamination effect of tissue
Fruit, it is difficult to obtain preferable clearly institutional framework.It is for group of shaping to the negative pressure of sample, fixation and embedding treatment in the present invention
Knit, extension tissue avoids fold overlapping, avoid the occurrence of because watery tissue elasticity error be present.The present invention it is easy to operate,
Feasibility is strong, the cycle is short, has well for the Shrimp waste histoorgan of the fixation of tissue morphology difficulty, soft texture, watery
Tissue area indexes, it is possible to achieve the paraffin section observation of complete tissue morphology.With the gill tissue of Macrobrachium nipponensis and hepatopancrease (in
Enteraden) exemplified by tissue, the light microscopic microexamination of paraffin section is carried out with the present invention, it can be clearly seen that：(1) gill tissue,
Vertical section gill leaf marshalling, close structure；Radial chrysanthemum structure is presented in the cross section branchial gland, and nephrocyte arrangement has rule
Rule property, is taper.(2) hepatopancrease (mid intestinal gland) tissue, liver tubule vertical section marshalling, cross section is star-shaped, clear in structure
Completely；Vertical section ductility is good, and tissue is substantial full, meets normal physiological form, and tissue is sprawled clear, non-overlapping.With this
Invention carries out histotomy to Shrimp waste watery tissue, can meet whole requirements that researcher is observed biopsy tissues.
Brief description of the drawings：
Fig. 1 is the cross-sectional slice of Macrobrachium nipponensis gill tissue and vertical section section in the embodiment of the present invention 1；
Fig. 2 is that the cross-sectional slice of Macrobrachium nipponensis hepatopancrease (mid intestinal gland) tissue and vertical section are cut in the embodiment of the present invention 1
Piece；
Fig. 3 is Fig. 1 colored schematic diagram；
Fig. 4 is Fig. 2 colored schematic diagram；
Fig. 1 with Fig. 2 method therefors are consistent, and simply sample tissue is different.Fig. 1 and Fig. 2 explanations carry out paraffin with this method
The light microscopic microexamination of section, it can be clearly seen that：(1) gill tissue, vertical section gill leaf marshalling, close structure；It is crosscutting
Radial chrysanthemum structure is presented in the face branchial gland, and nephrocyte arrangement has regularity, is taper.(2) hepatopancrease (mid intestinal gland) group
Knit, liver tubule vertical section marshalling, cross section is star-shaped, clear in structure complete；Vertical section ductility is good, organizes to enrich and satisfies
It is full, meet normal physiological form, tissue is sprawled clear, non-overlapping.
Embodiment：
Following embodiments are only described in further detail to the present invention, but do not form any limitation of the invention.
Embodiment 1
The paraffin section method of Shrimp waste watery tissue has been substantially carried out in the present embodiment, have chosen the gill of Macrobrachium nipponensis
Tissue and hepatopancrease (mid intestinal gland) tissue, are embodied by taking gill tissue as an example, the same gill tissue of hepatic tissue processing, including following
Step：
1. draw materials and fixed
2.5% paraformaldehyde solution is prepared in fume hood：With paraformaldehyde 50g, 1 × PBS cushioning liquid (Na2HPO4
8mM, NaCl 136mM, KH2PO42mM, KCl 2.6mM, PH=7.2-7.4) 900ml, 1000ml is added water to, 60 DEG C after mixing
Lower water-bath 24h, if there is paraformaldehyde to also have the NaOH solution of the undissolved equivalent of dropwise addition 1-2 drops 1, (10g powder is dissolved in 250ml water
The mixed solution of middle formation), regulation PH is 6.8-7.2, is preserved at 4 DEG C.
Lightly the carapace of Macrobrachium nipponensis is removed first, the 4th, 5 gill is removed to the 10ml band plugs for being put into cleaning
In clear glass ampoule, 2.5% appropriate paraformaldehyde solution is added, covers tightly plug so that negative-pressure container total measurement (volume) (V1)：
Fixer cumulative volume (V in container2):Fixing organization volume (V3)=6:3:1, lid is sealed, is taken out using 5ml disposable syringes
Gas formation -1.25kpa negative pressure, persistently handles 2-3min, until sample tissue surface bubble-free produces.Then gill tissue is connected
Poured into together with paraformaldehyde in 15ml centrifuge tube and add 2.5% paraformaldehyde to cover lid to scale 10ml, write mark
Label, are placed vertically in rack for test tube.Regular time is 4-6h, is usually no more than 24h.
Sample tissue is put in tissue fixative solution, negative pressure is fixed immediately, set time t >=5min, then is added to immediately
In the centrifuge tube for filling the appropriate volume of the tissue fixative solution of same concentrations, room temperature (20-25 DEG C) constant temperature fixes 4-6h；
2. flowing water rinses
The fixer remained in gill leaf texture sample blocks is removed with flowing clear water.Tissue is wrapped with lens wiping paper and is placed on plastic bag
Bury in box, marked sample tissue title and numbering, be put into the beaker of 2000ml capacity with the distilled water flushing 24h of flowing.
3. tissue dewatering
Liquidate and wash complete gill leaf texture sample and to carry out serial dehydration processing, the concentration of alcohol used in dehydration
Will step by step gradient it is progressive, slowly, be advisable in order；The volume ratio of ethanol solution and tissue block is 50:1 (≤70%) and 35:1 (＞
70%)；Under normal temperature (25 DEG C or so), tissue dewatering step is：
1) 25 DEG C, 4h is persistently handled in the ethanol of 30% concentration；
2) 25 DEG C, 40min is persistently handled in the ethanol of 40% concentration；
3) 25 DEG C, 40min is persistently handled in the ethanol of 50% concentration；
4) 25 DEG C, 40min is persistently handled in the ethanol of 60% concentration；
5) 25 DEG C, 40min is persistently handled in the ethanol of 70% concentration；
6) 25 DEG C, 40min is persistently handled in the ethanol of 80% concentration；
7) 25 DEG C, 40min is persistently handled in the ethanol of 90% concentration；
8) 25 DEG C, 40min is persistently handled in the ethanol of 95% concentration；
9) 25 DEG C, (I) 15min is persistently handled in the ethanol of 100% concentration；
10) 25 DEG C, (II) 15min is persistently handled in the ethanol of 100% concentration.
4. transparency of organization
With the solution of dimethylbenzene I (Solute mass point after embedded box equipped with gill leaf texture block is taken out from absolute ethyl alcohol
Number for 50%), the solution of dimethylbenzene II (Solute mass fraction 95%) carry out transparent processing successively, xylene solution and gill leaf texture
The volume ratio of block is 30:1 (I solution) and 15:1 (II solution).Transparency of organization step is：
1) exert oneself whipping arm, net residual ethanol solution is got rid of, to be dripped after hanging Action of Gravity Field 30s without ethanol solution drop
For standard；
2) 40min in the solution of dimethylbenzene I；
3) 30min in the solution of dimethylbenzene II.
5. ooze wax embedding
Pour paraffin into funnel to be filtered, transparent good tissue is then sequentially placed into wax pan, wax 1h is oozed at 60 DEG C, mould
Expect embedded box sequence, in case tissue is not inconsistent with label.Ooze and embedded after the completion of wax, put metal embedding device well, light
Alcolhol burner, the paraffin filtered is poured into wax kettle and is then filled with metal embedding device, sharp mouth tweezers burn on alcolhol burner, fast
The tissue for having oozed wax is put into metal embedding device by speed, puts the plastic embedding for the tissue being indicated after good position corresponding label
Box is gently put into metal embedded box slowly extruding and is laid flat, until wax is extruded from the hole of plastic embedding box, by plastic embedding box
Bottom embeds, and removes embedding device after complete cooled and solidified, preserved at next step experiment, or 4 DEG C.
6. section exhibition piece
Tighten and fix slicer head, consolidate cutter frame structure, adjustment slice thickness is 5 μm.That is cut into slices first is pre-
Experiment finishing, until after cutting out complete maximum tissue section, then carry out formal cutting.It is with tweezers that the wax disk(-sc) one end cut is careful
It is careful to lift, will cut into slices as far as possible it is smooth spread out, gently drag make its individual layer be laid on exhibition piece machine thermostatted water surface layer.Naturally dry 12h,
100 DEG C of oven for baking 2h.
7. dewaxing and HE dyeing
Dry section is taken, is put in dewaxing 10min in the dye vat for filling the solution of dimethylbenzene II (95%).HE dyeing
Step is：
1) 25 DEG C, 100% ethanol solution persistently handles 10min；
2) 25 DEG C, 95% ethanol solution persistently handles 10min；
3) 25 DEG C, 90% ethanol solution persistently handles 10min；
4) 25 DEG C, 80% ethanol solution persistently handles 10min；
5) 25 DEG C, 70% ethanol solution persistently handles 10min；
6) 25 DEG C, 60% ethanol solution persistently handles 10min；
7) 25 DEG C, distilled water persistently handles 5min, 4 DEG C of cooling 3min；
8) 25 DEG C, haematine dye liquor persistently handles 10min, 4 DEG C of cooling 3min；
9) 25 DEG C, distilled water is gently stained with 1-2 times, 4 DEG C of cooling 1-2min；
10) 25 DEG C, color separation liquid (70% alcohol 100ml, 0.5% hydrochloric acid 1ml) persistently handles 5s, 4 DEG C of cooling 10s；
11) 25 DEG C, deionized water is gently stained with 1-2 times, 4 DEG C of cooling 3min；
12) 25 DEG C, eosin stain persistently handles 1min, 4 DEG C of cooling 10s；
13) 25 DEG C, 90% ethanol solution persistently handles 2min；
14) 25 DEG C, 95% ethanol solution persistently handles 2min；
15) 25 DEG C, 100% ethanol solution persistently handles 10min；
16) 25 DEG C, dimethylbenzene I (50%) solution persistently handles 10min；
17) 25 DEG C, dimethylbenzene II (95%) solution persistently handles 10min.
Then, the section for dyeing completion is subjected to mounting processing with neutral gum；It will finally be sealed with 35 DEG C of insulating boxs
Drying process is made in section.
8. microexamination
In the case where Leica DM-2500 are just putting 40 times, 100 times, 200 times, 400 times of fluorescence microscope, tissues observed section.
Observation result is shown：Paraffin section after being handled with the inventive method, can be clear by light microscopic microexamination
See on ground：(1) gill tissue, vertical section gill leaf marshalling, close structure；Radial chrysanthemum structure is presented in the cross section branchial gland,
Nephrocyte arrangement has regularity, is taper.(2) hepatopancrease (mid intestinal gland) tissue, liver tubule vertical section marshalling are crosscutting
Face is star-shaped, clear in structure complete；Vertical section ductility is good, and tissue is substantial full, meets normal physiological form, and tissue is sprawled clear
It is clear, non-overlapping.
The present invention it is easy to operate, feasibility is strong, the cycle is short, for the fixation of tissue morphology difficulty, soft texture, watery shrimp
There is crab class loading organ good tissue area to index, it is possible to achieve the paraffin section observation of complete tissue morphology.With Japan
Exemplified by the gill tissue of pond crayfish and hepatopancrease (mid intestinal gland) tissue, the light microscopic microexamination of paraffin section is carried out with the present invention, can
Can be clearly seen that：(1) gill tissue, vertical section gill leaf marshalling, close structure；Radial chrysanthemum is presented in the cross section branchial gland
Structure, nephrocyte arrangement have regularity, are taper.(2) hepatopancrease (mid intestinal gland) tissue, the arrangement of liver tubule vertical section are whole
Together, cross section is star-shaped, clear in structure complete；Vertical section ductility is good, and tissue is substantial full, meets normal physiological form, group
Knit sprawl it is clear, non-overlapping.
Claims (5)
1. a kind of paraffin section method of Shrimp waste tissue, it is characterised in that step is as follows：
1) sample tissue is put in tissue fixative solution, negative pressure is fixed immediately, negative pressure to sample tissue block expand it is full, no longer produce
Anger bubble；It is added in the centrifuge tube of the appropriate volume for the tissue fixative solution for filling same concentrations, is organized in centrifuge tube immediately again
The ratio between amount and sample tissue volume of fixer are 20：1；Tissue block is avoided to be affixed on chamber wall, room temperature constant temperature fixes 4-6h；Institute
The pressure value for stating negative pressure is -1.25kpa；Negative-pressure container total measurement (volume)：Tissue fixative solution cumulative volume in container:Sample tissue volume=
6:3:1；
The tissue fixative solution is the paraformaldehyde solution of Solute mass fraction 2.5%；The specific preparation method of paraformaldehyde solution
For：Paraformaldehyde 50g, 1 × PBS cushioning liquid 900ml is taken, adds water to 1000ml, water-bath 24h at 60 DEG C after mixing, if also having
Undissolved paraformaldehyde, the NaOH solution of the equivalent of 1-2 drops 1 is added dropwise, regulation PH is 6.8-7.2, is preserved at 4 DEG C；
The component of 1 × PBS cushioning liquid is described in 0.01M：Na2HPO48mM, NaCl 136mM, KH2PO42mM, KCl
2.6mM, PH=7.2-7.4；
The NaOH solution of 1 equivalent is：Weigh NaOH solid powder 10g, with 250mL volumetric flask be configured to 250mL,
1mol/L NaOH solution.
2) sample tissue of the step 1) after fixed is put into embedded box, with the flowing uninterrupted flow wash embedded box 24h of clear water；
Remove the fixer remained in sample tissue；
3) liquidate and wash complete sample tissue at normal temperatures, successively with gradient concentration be 30%, 40%, 50%, 60%, 70%,
80%th, 90%, 100% ethanol solution dewater treatment；The ethanol solution dehydration treatment time that concentration is 30% is respectively 4h-
5h；The ethanol solution dehydration treatment time that concentration is 40%, 50%, 60%, 70% is respectively 40min-1h, concentration 80%,
90%th, 100% ethanol dehydration processing time is respectively 15min-35min；When ethanol solution concentration≤70%, the ethanol
The volume ratio of solution and sample tissue is 50:1；When ethanol solution concentration concentration >=70%, the ethanol solution and sample sets
The volume ratio knitted is 35:1；The embedded box equipped with sample tissue is taken out from ethanol solution after the completion of dewater treatment；
4) embedded box taken out in step 3) is put into clarifier and carries out transparent processing；
5) transparent good sample tissue is put into wax pan, wax 1h is oozed at 60 DEG C；Ooze and wrapped with metal embedding device after the completion of wax
Bury, remove embedding device after complete cooled and solidified；
6) tighten and fix slicer head, consolidate cutter frame structure, adjustment slice thickness is 5 μm；The pre- reality cut into slices first
Running repair is whole, until after cutting out complete maximum tissue section, then carry out formal cutting；To cut into slices it is smooth spread out, gently dragging makes its list
Layer is laid on the thermostatted water surface layer of exhibition piece machine；Naturally dry 12h, 100 DEG C of oven for baking 2h；
7) section after drying is subjected to dewaxing treatment with xylene solution, after the completion of dewaxing with 100%, 95%, 90%,
80%th, 70%, 60% concentration gradient ethanol solution and Yihong-haematine dye liquor carry out dyeing processing；Finally, neutral gum is used
The section progress mounting processing completed will be dyed, and drying process is made into the section sealed with insulating box.
A kind of 2. paraffin section method of Shrimp waste tissue as claimed in claim 1, it is characterised in that group in the step 3)
Knitting dehydration is：
1) 25 DEG C, 4h is persistently handled in the ethanol of 30% concentration；
2) 25 DEG C, 40min is persistently handled in the ethanol of 40% concentration；
3) 25 DEG C, 40min is persistently handled in the ethanol of 50% concentration；
4) 25 DEG C, 40min is persistently handled in the ethanol of 60% concentration；
5) 25 DEG C, 40min is persistently handled in the ethanol of 70% concentration；
6) 25 DEG C, 40min is persistently handled in the ethanol of 80% concentration；
7) 25 DEG C, 40min is persistently handled in the ethanol of 90% concentration；
8) 25 DEG C, 40min is persistently handled in the ethanol of 95% concentration；
9) 25 DEG C, (I) 15min is persistently handled in the ethanol of 100% concentration；
10) 25 DEG C, (II) 15min is persistently handled in the ethanol of 100% concentration.
3. a kind of paraffin section method of Shrimp waste tissue as claimed in claim 1, it is characterised in that in the step 4) thoroughly
Bright dose be Solute mass fraction be 50% the solution of dimethylbenzene I, Solute mass fraction be 95% the solution of dimethylbenzene II；
Transparent processing step is as follows：
1) net remaining embedded box ethanol solution is got rid of, to be dripped after hanging Action of Gravity Field 30s without ethanol solution drop as standard；
2) embedded box is first put into the solution of dimethylbenzene I and 30min in the solution of dimethylbenzene II is placed into after 40min.
A kind of 4. paraffin section method of Shrimp waste tissue as claimed in claim 1, it is characterised in that the preliminary experiment finishing
It is carried out in three steps：First, section, the section that each knife is cut out all is the monolithic histotomy of 5 μm of uniform thickness, is avoided the occurrence of
Uneven thickness；Second, cutting size, the shape of cross section for the section that each knife is cut out is similar with area；Third, cutting microstructure, lead to
To cross and constantly observed section under the microscope, the section microstructure for ensureing to cut out should meet crosscutting, rip cutting requirement,
It is simultaneously high-visible.
5. a kind of paraffin section method of Shrimp waste tissue as claimed in claim 1, it is characterised in that taken off in the step 7)
Wax step is：Dry section is taken, is put in and fills Solute mass fraction to be dewaxed in 95% solution of dimethylbenzene II
10min；
It is as follows to dye processing step：
1) 25 DEG C, 100% ethanol solution persistently handles 10min；
2) 25 DEG C, 95% ethanol solution persistently handles 10min；
3) 25 DEG C, 90% ethanol solution persistently handles 10min；
4) 25 DEG C, 80% ethanol solution persistently handles 10min；
5) 25 DEG C, 70% ethanol solution persistently handles 10min；
6) 25 DEG C, 60% ethanol solution persistently handles 10min；
7) 25 DEG C, distilled water persistently handles 5min, 4 DEG C of cooling 3min；
8) 25 DEG C, haematoxylin dye liquor persistently handles 10min, 4 DEG C of cooling 3min；
9) 25 DEG C, distilled water is gently stained with 1-2 times, 4 DEG C of cooling 1-2min；
10) 25 DEG C, color separation liquid persistently handles 5s, 4 DEG C of cooling 10s；11) 25 DEG C, deionized water is gently stained with 1-2 times, 4 DEG C of coolings
3min；The color separation liquid component is：The alcohol 100ml and hydrochloric acid 1ml of Solute mass fraction 70%；
12) 25 DEG C, eosin stain persistently handles 1min, 4 DEG C of cooling 10s；
13) 25 DEG C, 90% ethanol solution persistently handles 2min；
14) 25 DEG C, 95% ethanol solution persistently handles 2min；
15) 25 DEG C, 100% ethanol solution persistently handles 10min；
16) 25 DEG C, the solution of dimethylbenzene I that Solute mass fraction is 50% persistently handles 10min；
17) 25 DEG C, the solution of dimethylbenzene II that Solute mass fraction is 50% persistently handles 10min.
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| CN109580635A (en) * | 2018-12-03 | 2019-04-05 | 高佳太阳能股份有限公司 | A kind of diamond cut silicon wafer piece thickness ununiformity reason fast determination method |
| CN110887719A (en) * | 2019-12-20 | 2020-03-17 | 云南中医药大学 | Method for processing tissue sample of histopathology |
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