CN107389412B - A kind of paraffin section production method of Shrimp waste watery tissue - Google Patents
A kind of paraffin section production method of Shrimp waste watery tissue Download PDFInfo
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- CN107389412B CN107389412B CN201710768140.7A CN201710768140A CN107389412B CN 107389412 B CN107389412 B CN 107389412B CN 201710768140 A CN201710768140 A CN 201710768140A CN 107389412 B CN107389412 B CN 107389412B
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- 239000002699 waste material Substances 0.000 title claims abstract description 17
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- 238000000034 method Methods 0.000 claims abstract description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 229
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- 235000019441 ethanol Nutrition 0.000 claims description 90
- 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
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- 239000001993 wax Substances 0.000 claims description 15
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- 238000006297 dehydration reaction Methods 0.000 claims description 12
- 239000000834 fixative Substances 0.000 claims description 11
- 238000004043 dyeing Methods 0.000 claims description 8
- 239000012153 distilled water Substances 0.000 claims description 7
- 239000007788 liquid Substances 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
- 239000002184 metal Substances 0.000 claims description 6
- 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 description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
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- 230000005484 gravity Effects 0.000 claims description 3
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- 238000001574 biopsy Methods 0.000 abstract description 2
- 210000001519 tissue Anatomy 0.000 description 87
- 229960004756 ethanol Drugs 0.000 description 55
- 241000238557 Decapoda Species 0.000 description 14
- 210000000514 hepatopancreas Anatomy 0.000 description 9
- 210000004347 intestinal mucosa Anatomy 0.000 description 8
- 241000058338 Macrobrachium nipponense Species 0.000 description 5
- 230000008520 organization Effects 0.000 description 5
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- 230000007812 deficiency Effects 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 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
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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
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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
- G01N2001/2873—Cutting or cleaving
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- Physics & Mathematics (AREA)
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Abstract
The present invention relates to a kind of paraffin section production methods of Shrimp waste watery tissue；Conventional paraffin tissue sections method index is for the fixation of tissue morphology difficulty, soft, watery the inorganizable discrimination of Shrimp waste histoorgan, easily cause historrhexis, overlapping, warpage, seriously affect the microexamination effect of tissue, it is difficult to obtain ideal clearly institutional framework.Be formed and then negative pressure to sample, fixation and embedding treatment tissue, extension tissue of the present invention avoids that fold is overlapped and there are errors due to the elasticity of watery tissue.Operation of the present invention is easy, feasibility is strong, the period is short, the fixation of tissue morphology difficulty, soft, watery Shrimp waste histoorgan are indexed with good tissue area, the paraffin section observation that complete tissue morphology may be implemented is able to satisfy whole requirements that researcher observes biopsy tissues.
Description
Technical field
The present invention relates to a kind of paraffin section production methods of Shrimp waste watery tissue, belong to microscopic tissue sections technology
Field.
Background technique:
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 various histoorgans is higher, especially through frequently as pathological research foundation
Gill tissue and hepatopancrease (mid intestinal gland) tissue.In recent years, to Shrimp waste both the above organize in research process materials amount it is big,
Reagent dosage is more, and for economic consideration, traditional technology is still used as and technology is widely used.Technology generally comprise materials and fixation,
Flowing water flushing, transparency of organization, seeps wax embedding, slice exhibition piece, dewaxing and HE dyeing, light microscopic microexamination at tissue dewatering.
And conventional method index is for the fixation of tissue morphology difficulty, soft, watery the inorganizable area of Shrimp waste histoorgan
Indexing, easily causes historrhexis, overlapping, warpage, seriously affects the microexamination effect of tissue, it is difficult to obtain ideal clearly group
Knit structure.The present invention is directed to make up existing method in the waterys animals such as processing Shrimp waste and its technical deficiency of tissue, to obtain
Obtain ideal histoorgan paraffin section image.
Summary of the invention:
To solve the above-mentioned problems, the present invention provides a kind of paraffin section production methods of Shrimp waste watery tissue.
A kind of paraffin section production method of Shrimp waste tissue, steps are as follows:
1, 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 generated again；It is added to immediately in the centrifuge tube for the appropriate volume of tissue fixative solution for filling same concentrations again, 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 are as follows: take paraformaldehyde 50g, 1 × PBS buffer solution 900ml to add water to 1000ml, after mixing at 60 DEG C water-bath for 24 hours, if
There are also undissolved paraformaldehydes, and the NaOH solution of 1 equivalent of 1-2 drop (1mol/L) is added dropwise, and adjusting PH is 6.8-7.2, protect at 4 DEG C
It deposits；
The component of 1 × PBS buffer solution (0.01M) are as follows: Na2HPO48mM, NaCl 136mM, KH2PO42mM,
KCl 2.6mM, PH=7.2-7.4；
The NaOH solution of 1 equivalent (1mol/L) are as follows: weigh NaOH solid powder 10g, configured with the volumetric flask of 250mL
At the NaOH solution of 250mL, 1mol/L.
2, 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 remaining fixer in sample tissue；It is wrapped after flushing with lens wiping paper.
3, liquidate and wash complete sample tissue under room temperature (25 DEG C or so), successively with gradient concentration be 30%, 40%,
50%, 60%, 70%, 80%, 90%, 100% ethanol solution dehydration；The ethanol solution dehydration that concentration is 30%
Time is respectively 4h-5h；The ethanol solution dehydration treatment time that concentration is 40%, 50%, 60%, 70% is respectively 40min-
The ethanol dehydration processing time of 1h, concentration 80%, 90%, 100% are 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 ethyl alcohol 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 dehydration
Middle taking-up.
4, the 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, seeps wax 1h at 60 DEG C.Seep wax after the completion of use metal embed device into
Row embedding removes embedding device after complete cooled and solidified, save at next step test or 4 DEG C.
6, it tightens and fixes slicer head, consolidate cutter frame structure, adjustment slice thickness is 5 μm.It is sliced first
Preliminary experiment finishing, until after cutting out complete maximum tissue section, then carry out formal cutting.To be sliced as far as possible it is smooth spread out, gently
Drag the thermostatted water surface layer for making its single 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 is that tangent plane, guarantees that the slice that each knife is cut out all is 5 μm uniformly thick
The monolithic histotomy of degree, avoids the occurrence of uneven thickness；Second is that cutting size, guarantee the cross-sectional shape for the slice that each knife is cut out
It is similar with area；Third is that cutting microstructure, is constantly observed under the microscope by that will be sliced, guarantee that the slice cut out is microcosmic
Tissue should meet crosscutting, longitudinal sectional requirement, while high-visible.
7, the slice after drying is subjected to dewaxing treatment with dimethylbenzene II solution, after the completion of dewaxing with 100%, 95%,
90%, 80%, 70%, 60% concentration gradient ethanol solution and Yihong-hematoxylin dye liquor carry out dyeing processing；Finally, in
Property the natural gum slice of completing dyeing carry out mounting processing, and the slice sealed is dried with insulating box.
Preferably, tissue dewatering step in the step 3) are as follows:
1) 25 DEG C, 4h is persistently handled in the ethyl alcohol of 30% concentration；
2) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 40% concentration；
3) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 50% concentration；
4) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 60% concentration；
5) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 70% concentration；
6) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 80% concentration；
7) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 90% concentration；
8) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 95% concentration；
9) 25 DEG C, 15min is persistently handled in the ethyl alcohol of 100% concentration；
10) 25 DEG C, 15min is persistently handled in the ethyl alcohol of 100% concentration.
In the step 4) clarifier be Solute mass fraction be 50% I solution of dimethylbenzene, Solute mass fraction be
95% II solution of dimethylbenzene.I solution component of dimethylbenzene is that 100% ethyl alcohol and 100% dimethylbenzene are mixed with volume ratio 1:1
It closes；II solution component of dimethylbenzene is that 100% ethyl alcohol is mixed with 100% dimethylbenzene with volume ratio 1:19.
Transparency of organization processing step in the step 4) are as follows:
1) get rid of net remaining embedded box ethanol solution, with after hanging gravity 30s without ethanol solution drop drip for
Standard；
2) embedded box is first put into I solution of dimethylbenzene and places into 30min in II solution of dimethylbenzene after 40min.
Hydrodewaxing step in the step 7) are as follows: take dried slice, being put in and filling Solute mass fraction is 95%
Dewax 10min in II solution of dimethylbenzene.
In the step 7) the step of dyeing processing are as follows:
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, hematoxylin 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 are as follows: the alcohol of Solute mass fraction 70% and 0.5% hydrochloric acid 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, I solution of dimethylbenzene that Solute mass fraction is 50% persistently handles 10min；
17) 25 DEG C, II solution of dimethylbenzene that Solute mass fraction is 95% persistently handles 10min.
The invention has the advantages that:
Currently, conventional paraffin tissue sections method index is for the fixation of tissue morphology difficulty, soft, watery shrimp
The inorganizable discrimination of crab class loading organ, easily causes historrhexis, overlapping, warpage, seriously affects the microexamination effect of tissue
Fruit, it is difficult to obtain ideal clearly institutional framework.It is for group of being formed to the negative pressure of sample, fixation and embedding treatment in the present invention
Knit, extension tissue avoids fold from being overlapped, avoid the occurrence of due to the elasticity of watery tissue that there are errors.Operation of the present invention simplicity,
Feasibility is strong, the period is short, has for the fixation of tissue morphology difficulty, soft, watery Shrimp waste histoorgan good
The paraffin section observation of complete tissue morphology may be implemented in tissue area indexing.With the gill tissue of Macrobrachium nipponensis and hepatopancrease (in
Enteraden) for tissue, the light microscopic microexamination of paraffin section is carried out with the present invention, it can be clearly seen that: (1) gill tissue,
Longitudal 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 longitudal section marshalling, cross section is star-shaped, clear in structure
Completely；Longitudal section ductility is good, and tissue enriches full, meets normal physiological form, tissue is sprawled clear, non-overlapping.With this
Invention carries out histotomy to Shrimp waste watery tissue, can satisfy whole requirements that researcher observes biopsy tissues.
Detailed description of the invention:
Fig. 1 is cross-sectional slice and the longitudal section slice of Macrobrachium nipponensis gill tissue 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 longitudal section are cut in the embodiment of the present invention 1
Piece；
Fig. 3 is the colored schematic diagram of Fig. 1；
Fig. 4 is the colored schematic diagram of Fig. 2；
Fig. 1 with Fig. 2 method therefor is consistent, and only sample tissue is different.Fig. 1 and Fig. 2 explanation carries out paraffin with this method
The light microscopic microexamination of slice, it can be clearly seen that: (1) gill tissue, longitudal section gill leaf marshalling, close structure；It is crosscutting
Radial chrysanthemum structure is presented in the face branchial gland, and it is taper that nephrocyte arrangement, which has regularity,.(2) hepatopancrease (mid intestinal gland) group
It knits, liver tubule longitudal section marshalling, cross section is star-shaped, clear in structure complete；Longitudal section ductility is good, organizes to enrich full
It is full, meet normal physiological form, tissue is sprawled clear, non-overlapping.
Specific embodiment:
Only invention is further described in detail for following embodiments, but does not constitute any limitation of the invention.
Embodiment 1
It has been substantially carried out the paraffin section method of Shrimp waste watery tissue in the present embodiment, has had 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. drawing materials and fixed
2.5% paraformaldehyde solution is prepared in draught cupboard: with paraformaldehyde 50g, 1 × PBS buffer solution (Na2HPO4
8mM, NaCl 136mM, KH2PO42mM, KCl 2.6mM, PH=7.2-7.4) 900ml, 1000ml is added water to, 60 DEG C after mixing
For 24 hours, if there is paraformaldehyde there are also the undissolved NaOH solution that 1 equivalent of 1-2 drop is added dropwise, (10g powder is dissolved in 250ml water for lower water-bath
The mixed solution of middle formation), adjusting PH is 6.8-7.2, is saved at 4 DEG C.
Lightly the carapace of Macrobrachium nipponensis is removed first, the 4th, 5 gill is removed and is put into clean 10ml band rubber plug
In transparent glass ampoule, suitable 2.5% paraformaldehyde solution is added, rubber plug is covered tightly, so that negative-pressure container total measurement (volume) (V1):
Fixer total volume (V in container2): fixing organization volume (V3)=6:3:1, seals lid, is taken out using 5ml disposable syringe
The negative pressure of gas formation -1.25kpa, persistently handles 2-3min, until sample tissue surface bubble-free generates.Then gill tissue is connected
It is poured into together with paraformaldehyde in the centrifuge tube of 15ml and adds 2.5% paraformaldehyde to scale 10ml, covered lid, write mark
Label, are placed vertically in rack for test tube.Regular time is 4-6h, is usually no more than for 24 hours.
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, the fixed 4-6h of room temperature (20-25 DEG C) constant temperature；
2. flowing water rinses
Remaining fixer 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
It buries in box, has marked sample tissue title and number, be put into the beaker of 2000ml capacity with the distilled water flushing of flowing for 24 hours.
3. tissue dewatering
It liquidates and washes complete gill leaf texture sample and to carry out serial dehydration processing, concentration of alcohol used in dehydration
Will step by step gradient it is progressive, slowly, be orderly advisable；The volume ratio of ethanol solution and tissue block is 50:1 (≤70%) and 35:1 (>
70%)；Under room temperature (25 DEG C or so), tissue dewatering step are as follows:
1) 25 DEG C, 4h is persistently handled in the ethyl alcohol of 30% concentration；
2) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 40% concentration；
3) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 50% concentration；
4) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 60% concentration；
5) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 70% concentration；
6) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 80% concentration；
7) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 90% concentration；
8) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 95% concentration；
9) 25 DEG C, (I) 15min is persistently handled in the ethyl alcohol of 100% concentration；
10) 25 DEG C, (II) 15min is persistently handled in the ethyl alcohol of 100% concentration.
4. transparency of organization
By the embedded box equipped with gill leaf texture block with I solution of dimethylbenzene (Solute mass point after being taken out in dehydrated alcohol
Number for 50%), II solution of dimethylbenzene (Solute mass fraction 95%) successively carry out transparent processing, 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 are as follows:
1) it exerts oneself whipping arm, net residual ethanol solution is got rid of, to drip after hanging gravity 30s without ethanol solution drop
For standard；
2) 40min in I solution of dimethylbenzene；
3) 30min in II solution of dimethylbenzene.
5. seeping wax embedding
It pours paraffin into funnel to be filtered, transparent good tissue is then sequentially placed into wax pan, wax 1h, modeling are seeped at 60 DEG C
Expect embedded box sequence, in case tissue is not inconsistent with label.It seeps wax to be embedded after the completion, puts metal embedding device well, light
Filtered paraffin is poured into wax pot and is then filled with metal embedding device by alcolhol burner, and sharp mouth tweezers are burnt on alcolhol burner, fast
The tissue for having seeped 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 to squeeze and be laid flat, until wax is squeezed out from the hole of plastic embedding box, by plastic embedding box
Bottom embeds, and removes embedding device after complete cooled and solidified, save at next step test or 4 DEG C.
6. slice exhibition piece
It tightens and fixes slicer head, consolidate cutter frame structure, adjustment slice thickness is 5 μm.What is be sliced 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 be sliced as far as possible it is smooth spread out, gently drag make its single 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
Dried slice is taken, the 10min that dewaxes is put in the dye vat for filling II solution of dimethylbenzene (95%).HE dyeing
Step are as follows:
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, hematoxylin 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 slice that dyeing is completed is subjected to mounting processing with neutral gum；It will finally be sealed with 35 DEG C of insulating boxs
Slice is dried.
8. microexamination
In the case where Leica DM-2500 is just setting 40 times, 100 times, 200 times, 400 times of fluorescence microscope, histotomy is observed.
Observation is as the result is shown: with the method for the present invention, treated that paraffin section can be clear by light microscopic microexamination
See on ground: (1) gill tissue, longitudal 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 longitudal section marshalling are crosscutting
Face is star-shaped, clear in structure complete；Longitudal section ductility is good, and tissue enriches full, meets normal physiological form, tissue is sprawled clear
It is clear, non-overlapping.
Operation of the present invention is easy, feasibility is strong, the period is short, for the fixation of tissue morphology difficulty, soft, watery shrimp
Crab class loading organ is indexed with good tissue area, and the paraffin section observation of complete tissue morphology may be implemented.With Japan
For 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, it can
Can be clearly seen that: (1) gill tissue, longitudal 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 longitudal section are whole
Together, cross section is star-shaped, clear in structure complete；Longitudal section ductility is good, and tissue enriches full, meets normal physiological form, group
Knit sprawl it is clear, non-overlapping.
Claims (1)
1. a kind of paraffin section method of Shrimp waste tissue, it is characterised in that steps are 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 for the appropriate volume of 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, the fixed 4-6h of room temperature constant temperature；Institute
The pressure value for stating negative pressure is -1.25kpa；Negative-pressure container total measurement (volume): tissue fixative solution total 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
Are as follows: take paraformaldehyde 50g, 1 × PBS buffer solution 900ml to add water to 1000ml, after mixing at 60 DEG C water-bath for 24 hours, if also
The NaOH solution of 1 equivalent of 1-2 drop (1mol/L) is added dropwise in undissolved paraformaldehyde, and adjusting PH is 6.8-7.2, saves at 4 DEG C；
The component of 1 × PBS buffer solution described in 0.01M are as follows: Na2HPO48mM, NaCl 136mM, KH2PO42mM, KCl
2.6mM, PH=7.2-7.4；
The NaOH solution of 1 equivalent are as follows: weigh NaOH solid powder 10g, with the volumetric flask of 250mL be configured to 250mL,
The NaOH solution of 1mol/L；
2) 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 remaining fixer in sample tissue；
3) liquidate and wash complete sample tissue at normal temperature, successively with gradient concentration be 30%, 40%, 50%, 60%, 70%, 80%,
90%, 100% ethanol solution dehydration；The ethanol solution dehydration treatment time that concentration is 30% is respectively 4h-5h；Concentration is
40%, 50%, 60%, 70% ethanol solution dehydration treatment time is respectively 40min-1h, the ethyl alcohol of concentration 80%, 90%, 100%
Dehydration treatment time is respectively 15min-35min；When ethanol solution concentration≤70%, the ethanol solution and sample tissue
Volume ratio is 50:1；When ethanol solution concentration concentration >=70%, the volume ratio of the ethanol solution and sample tissue is 35:1；
The embedded box equipped with sample tissue is taken out from ethanol solution after the completion of dehydration；
The tissue dewatering step are as follows:
A) 25 DEG C, 4h is persistently handled in the ethyl alcohol of 30% concentration；
B) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 40% concentration；
C) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 50% concentration；
D) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 60% concentration；
E) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 70% concentration；
F) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 80% concentration；
G) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 90% concentration；
H) 25 DEG C, 40min is persistently handled in the ethyl alcohol of 95% concentration；
I) 25 DEG C, (I) 15min is persistently handled in the ethyl alcohol of 100% concentration；
J) 25 DEG C, (II) 15min is persistently handled in the ethyl alcohol of 100% concentration；
4) embedded box taken out in step 3) is put into clarifier and carries out transparent processing；
The clarifier be Solute mass fraction be 50% I solution of dimethylbenzene, Solute mass fraction be 95% dimethylbenzene II it is molten
Liquid；
Steps are as follows for transparent processing:
A) net remaining embedded box ethanol solution is got rid of, to drip after hanging gravity 30s without ethanol solution drop as standard；
B) embedded box is first put into I solution of dimethylbenzene and places into 30min in II solution of dimethylbenzene after 40min；
5) transparent good sample tissue is put into wax pan, seeps wax 1h at 60 DEG C；Wax is seeped to be wrapped with metal embedding device after the completion
It buries, removes embedding device after complete cooled and solidified；
6) it tightens and fixes slicer head, consolidate cutter frame structure, adjustment slice thickness is 5 μm；The pre- reality being sliced first
Running repair is whole, until after cutting out complete maximum tissue section, then carry out formal cutting；To be sliced 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；
The preliminary experiment finishing is carried out in three steps: first is that tangent plane, the slice that each knife is cut out all is the monolithic of 5 μm of uniform thickness
Histotomy avoids the occurrence of uneven thickness；Second is that cutting size, the cross-sectional shape for the slice that each knife is cut out is similar with area；
Third is that cutting microstructure, constantly observed under the microscope by that will be sliced, guarantees that the slice microstructure cut out should expire
Crosscutting, the longitudinal sectional requirement of foot, at the same it is high-visible；
7) slice after drying is subjected to dewaxing treatment with xylene solution, after the completion of dewaxing with 100%, 95%, 90%, 80%,
70%, 60% concentration gradient ethanol solution and Yihong-hematoxylin dye liquor carry out dyeing processing；Finally, will be dyed with neutral gum
The slice of completion carries out mounting processing, and is dried the slice sealed with insulating box；
The hydrodewaxing step are as follows: take dried slice, be put in and fill in II solution of dimethylbenzene that Solute mass fraction is 95%
Dewax 10min；
It is as follows to dye processing step:
A) 25 DEG C, 100% ethanol solution persistently handles 10min；
B) 25 DEG C, 95% ethanol solution persistently handles 10min；
C) 25 DEG C, 90% ethanol solution persistently handles 10min；
D) 25 DEG C, 80% ethanol solution persistently handles 10min；
E) 25 DEG C, 70% ethanol solution persistently handles 10min；
F) 25 DEG C, 60% ethanol solution persistently handles 10min；
G) 25 DEG C, distilled water persistently handles 5min, 4 DEG C of cooling 3min；
H) 25 DEG C, haematoxylin dye liquor persistently handles 10min, 4 DEG C of cooling 3min；
I) 25 DEG C, distilled water is gently stained with 1-2 times, 4 DEG C of cooling 1-2min；
J) 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 cooling 3min；
The color separation liquid component are as follows: the alcohol 100ml and hydrochloric acid 1ml of Solute mass fraction 70%；
K) 25 DEG C, eosin stain persistently handles 1min, 4 DEG C of cooling 10s；
L) 25 DEG C, 90% ethanol solution persistently handles 2min；
M) 25 DEG C, 95% ethanol solution persistently handles 2min；
N) 25 DEG C, 100% ethanol solution persistently handles 10min；
O) 25 DEG C, I solution of dimethylbenzene that Solute mass fraction is 50% persistently handles 10min；
P) 25 DEG C, II solution of dimethylbenzene that Solute mass fraction is 50% persistently handles 10min.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101390510A (en) * | 2008-11-12 | 2009-03-25 | 中国农业大学 | Preprocessing method of Orthoptera insect integer serial section |
| CN103759994A (en) * | 2014-01-02 | 2014-04-30 | 河南科技大学 | Larva paraffin section forming method |
| CN105973673A (en) * | 2016-06-30 | 2016-09-28 | 中国林业科学研究院热带林业研究所 | Paraffin sectioning method for eucalyptus tissue |
| CN106644644A (en) * | 2016-12-30 | 2017-05-10 | 潍坊医学院 | Quick and low-toxic mosquito paraffin section manufacturing method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101390510A (en) * | 2008-11-12 | 2009-03-25 | 中国农业大学 | Preprocessing method of Orthoptera insect integer serial section |
| CN103759994A (en) * | 2014-01-02 | 2014-04-30 | 河南科技大学 | Larva paraffin section forming method |
| CN105973673A (en) * | 2016-06-30 | 2016-09-28 | 中国林业科学研究院热带林业研究所 | Paraffin sectioning method for eucalyptus tissue |
| CN106644644A (en) * | 2016-12-30 | 2017-05-10 | 潍坊医学院 | Quick and low-toxic mosquito paraffin section manufacturing method |
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