CN105928752B - A kind of insect imago paraffin section colouring method - Google Patents
A kind of insect imago paraffin section colouring method Download PDFInfo
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- CN105928752B CN105928752B CN201610226169.8A CN201610226169A CN105928752B CN 105928752 B CN105928752 B CN 105928752B CN 201610226169 A CN201610226169 A CN 201610226169A CN 105928752 B CN105928752 B CN 105928752B
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- 238000000034 method Methods 0.000 title claims abstract description 23
- 241000238631 Hexapoda Species 0.000 title claims abstract description 22
- 239000012188 paraffin wax Substances 0.000 title claims abstract description 14
- 238000004040 coloring Methods 0.000 title claims abstract description 8
- 238000004043 dyeing Methods 0.000 claims abstract description 7
- 230000008520 organization Effects 0.000 claims abstract description 6
- 230000018044 dehydration Effects 0.000 claims abstract description 5
- 238000006297 dehydration reaction Methods 0.000 claims abstract description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 46
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 27
- 235000019441 ethanol Nutrition 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 239000012153 distilled water Substances 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 13
- 239000011521 glass Substances 0.000 claims description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- 239000008096 xylene Substances 0.000 claims description 9
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 239000000834 fixative Substances 0.000 claims description 4
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 3
- ABBQHOQBGMUPJH-UHFFFAOYSA-M Sodium salicylate Chemical compound [Na+].OC1=CC=CC=C1C([O-])=O ABBQHOQBGMUPJH-UHFFFAOYSA-M 0.000 claims description 3
- 238000001574 biopsy Methods 0.000 claims description 3
- 230000004069 differentiation Effects 0.000 claims description 3
- 238000007654 immersion Methods 0.000 claims description 3
- 230000007935 neutral effect Effects 0.000 claims description 3
- 229920002866 paraformaldehyde Polymers 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 229960004025 sodium salicylate Drugs 0.000 claims description 3
- 238000007711 solidification Methods 0.000 claims description 3
- 230000008023 solidification Effects 0.000 claims description 3
- 238000004781 supercooling Methods 0.000 claims description 3
- 238000003860 storage Methods 0.000 claims description 2
- 210000000056 organ Anatomy 0.000 abstract description 7
- 210000002249 digestive system Anatomy 0.000 abstract description 3
- 239000012530 fluid Substances 0.000 abstract description 3
- 210000000653 nervous system Anatomy 0.000 abstract description 3
- 210000004994 reproductive system Anatomy 0.000 abstract description 3
- 210000002345 respiratory system Anatomy 0.000 abstract description 3
- 239000000463 material Substances 0.000 abstract description 2
- 238000013081 phylogenetic analysis Methods 0.000 abstract description 2
- 230000007812 deficiency Effects 0.000 abstract 1
- 239000000975 dye Substances 0.000 description 3
- 238000004821 distillation Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
-
- 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
- G01N1/36—Embedding or analogous mounting of samples
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
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Abstract
A kind of insect imago paraffin section colouring method, belong to microscopic tissue sections technical field, including tissue fixation, tissue dewatering, transparency of organization, waxdip, embedding and slice, exhibition piece and dry piece, dewaxing and tissue rehydration, dyeing, dehydration and transparent, mounting and observation, final slice indexes tissue area high, the space structure of each organ and tissue in polypide, the adult polypide material hard suitable for body wall, cavity fluids content is less can be obviously observed.Eye fly adult polypide is handled using this method, internal digestive system, excretory system, nervous system, respiratory system, the circulatory system and reproductive system can be clearly observed under the microscope.Insect imago entirety paraffin section method of the present invention can not only fill up the blank of insect imago internal and structure observation, can also make up the deficiency locally observed, and provide characteristic of division for insect Phylogenetic Analysis, easy to operate, time-consuming short, at low cost.
Description
Technical field
The present invention relates to a kind of insect imago paraffin section colouring methods, belong to microscopic tissue sections technical field, especially
It is to be related to a kind of insect imago paraffin section colouring method.
Background technique
Insect paraffin section and staining technique are to study the important experimental technique of insect tissue form and histopathology, generally
Including dissection, fixation, dehydration it is transparent, embedding, slice, dyeing and etc..Paraffin section and staining technique are chiefly used in insect at present
The local organization or organ of larva and adult, such as feeler, alimentary canal, foot.So not it is observed that whole inside insect imago
The space structure of organ and tissue.Internal's form of adult not only reflects its physiological characteristic, and due to internal structure compared with
Formalness is affected by environment small, usually more there is the stability of kind.But because adult ectoskeleton is hard, cavity fluids content is few,
Conventional dicing method is difficult to make complete paraffin section at present；And the dicing method of adult local organization or organ is not only grasped
Make complexity, time-consuming, and often needs the more advanced instrument and equipments such as negative pressure of vacuum processing, it is often more important that finally fixes, embeds, cutting
The treatment effect of piece etc. is not ideal enough, and final part slice indexes tissue area extremely limited, it is difficult to observe in complete polypide
The space structure of each system in portion and organ.
Summary of the invention
The object of the present invention is to provide a kind of insect imago paraffin section colouring method, the method is not only easy to operate, consumes
When it is short, at low cost, additionally it is possible to obviously observe the space structure of each organ and tissue in polypide.
The technical scheme adopted by the invention is that：
A kind of insect imago paraffin section colouring method, includes the following steps：
（1）Tissue is fixed：
It removes except the insect body of foot and wing is dipped in tissue fixative solution, stands 1 hour at room temperature, stood at 4 DEG C
12 hours；Tissue fixative solution is：The TritonX-100-4% paraformaldehyde mixed stationary liquid that concentration is 0.5%.Storage temperature is zero
Lower 20 DEG C, dosage and body volume ratio are 50:1.
（2）Tissue dewatering：
It will pass through（1）Treated polypide is taken out, and under the conditions of 4 DEG C, distilled water is rinsed, then at successively using at room temperature
The ethanol solution that concentration gradient is 70%, 80%, 90%, 95%, 100% is dehydrated, and the processing time of each gradient is 20 minutes；Distillation
Water and ethanol consumption and the volume ratio of polypide are 50:1.
（3）Transparency of organization：
It will pass through（2）Treated, and polypide is taken out, at room temperature successively in dimethylbenzene：Ethyl alcohol 1:3 mixed liquors, dimethylbenzene：Second
Alcohol 1:It impregnates 10 minutes in 1 mixed liquor, then is impregnated 5 minutes in pure xylene solution；
（4）Waxdip, embedding and slice；
It will pass through（3）Treated, and polypide is taken out, in 55 DEG C of atoleine：Dimethylbenzene 1:It is small that 2 are impregnated in 1 mixed liquor
When, it is impregnated 4 hours in 60 DEG C of atoleine, takes out, be sliced after supercooling, solidification, setting；
（5）It opens up piece and dries piece：
It taking slice to move to smear bonding die agent and be preheated on 55 DEG C of glass slide, slice is unfolded completely naturally immediately, then
12 hours baking pieces under the conditions of being placed in 37 DEG C；The group of bonding die agent becomes：Protein liquid, glycerol, sodium salicylate.Its ratio be 10:10:1.
（6）Dewaxing and tissue rehydration：
It takes the glass slide after drying piece to be immersed in xylene solution, is kept for 5 minutes, then successively soaking concentration gradient is
100%, 95%, 90%, 80%, 70% ethanol solution, then be soaked in distilled water, the processing time of each gradient is 1 ~ 3 minute.
（7）Dyeing：
Glass slide after taking tissue rehydration is immersed in hematoxylin solution, is kept for 5 minutes, is moved to and is rinsed 1 point in distilled water
Clock is then moved to and is returned blue liquid immersion 1 minute, 30 seconds in differentiation liquid, finally dyes 2 minutes in the solution of Yihong.
（8）Dehydration with it is transparent：
Take dyeing after glass slide carry out distilled water rinse 5 minutes, then be successively immersed in concentration gradient be 70%, 80%,
90%, it 2 ~ 5 minutes in 95%, 100% ethanol solution, moves in xylene solution and impregnates 3 minutes.
（9）Mounting and observation
Neutral gum is added dropwise on biopsy tissues, covered is placed in draught cupboard after being dried, under the microscope
Observation.
Beneficial effects of the present invention：
Insect imago paraffin section method in the present invention, easy to operate, time-consuming short, at low cost, final slice is to tissue area
Indexing is high, can obviously observe the space structure of each organ and tissue in polypide, hard, cavity fluids contain suitable for body wall
Measure less adult polypide material.Eye fly adult polypide is handled using this method, can be clearly observed under the microscope interior
Portion's digestive system, excretory system, nervous system, respiratory system, the circulatory system and reproductive system.Insect imago entirety stone of the present invention
Wax dicing method can not only fill up the blank of insect imago internal and structure observation, can also make up and locally observe not
Foot, provides characteristic of division for insect Phylogenetic Analysis.
Detailed description of the invention
Fig. 1 is female eye fly adult abdomen sectional view；
Fig. 2 is male eye fly adult integrally longitudinal sectional figure.
Specific embodiment
Only invention is further described in detail for following embodiments, but does not constitute any limitation of the invention.
Embodiment 1
It is eye fly Paraffin Sections of Adult Worm method in the present embodiment, includes the following steps：
1, tissue is fixed
The TritonX-100-4% paraformaldehyde mixed stationary liquid of compound concentration 0.5% in chemical hood.
Fixing process is：Examination is collected for polypide and is placed on sled, directly launches to mixing and fixes after removal foot and wing
In liquid, the amount and body volume ratio of fixer are 50：12 hours are stood at a temperature of Isosorbide-5-Nitrae DEG C；Test insect is artificial Intelligent culture
Raising eye fly adult in case, 26 DEG C of raising temperature, relative humidity 70%
2, tissue dewatering
It will pass through（1）Treated polypide is taken out, and under the conditions of 4 DEG C, distilled water is rinsed, then at successively using at room temperature
The ethanol solution that concentration gradient is 70%, 80%, 90%, 95%, 100% is dehydrated, and the processing time of each gradient is 20 minutes；Distillation
Water and ethanol consumption and the volume ratio of polypide are 50:1.
3, transparency of organization：
It will pass through（2）Treated, and polypide is taken out, at room temperature successively in dimethylbenzene：Ethyl alcohol 1:3 mixed liquors, dimethylbenzene：Second
Alcohol 1:It impregnates 10 minutes in 1 mixed liquor, then is impregnated 5 minutes in pure xylene solution；
4, waxdip, embedding and slice；
It will pass through（3）Treated, and polypide is taken out, in 55 DEG C of atoleine：Dimethylbenzene 1:It is small that 2 are impregnated in 1 mixed liquor
When, it is impregnated 4 hours in 60 DEG C of atoleine, takes out, be sliced after supercooling, solidification, setting；
5, exhibition piece and baking piece：
It taking slice to move to smear bonding die agent and be preheated on 55 DEG C of glass slide, slice is unfolded completely naturally immediately, then
12 hours baking pieces under the conditions of being placed in 37 DEG C；The group of bonding die agent becomes：Protein liquid, glycerol, sodium salicylate.Its ratio be 10:10:1.
6, dewax and organize rehydration：
It takes the glass slide after drying piece to be immersed in xylene solution, is kept for 5 minutes, then successively soaking concentration gradient is
100%, 95%, 90%, 80%, 70% ethanol solution, then be soaked in distilled water, the processing time of each gradient is 1 ~ 3 minute.
7, it dyes：
Glass slide after taking tissue rehydration is immersed in hematoxylin solution, is kept for 5 minutes, is moved to and is rinsed 1 point in distilled water
Clock is then moved to and is returned blue liquid immersion 1 minute, 30 seconds in differentiation liquid, finally dyes 2 minutes in the solution of Yihong.
8, dehydration with it is transparent：
Take dyeing after glass slide carry out distilled water rinse 5 minutes, then be successively immersed in concentration gradient be 70%, 80%,
90%, it 2 ~ 5 minutes in 95%, 100% ethanol solution, moves in xylene solution and impregnates 3 minutes.
9, mounting and observation
Neutral gum is added dropwise on biopsy tissues, covered is placed in draught cupboard after being dried, under the microscope
Observation.
It can be clearly observed internal digestive system under the microscope, excretory system, nervous system, respiratory system, follow
Loop system and reproductive system are shown in Fig. 1, Fig. 2.
Claims (1)
1. a kind of insect imago paraffin section colouring method, which is characterized in that the method includes following procedure：
(1) tissue is fixed：
It removes except the insect body of foot and wing is dipped in tissue fixative solution, stands 1 hour at room temperature, it is small that 12 are stood at 4 DEG C
When；Tissue fixative solution is：The TritonX-100-4% paraformaldehyde mixed stationary liquid that concentration is 0.5%；Storage temperature is subzero 20
DEG C, dosage and body volume ratio are 50:1；
（2）Tissue dewatering：
It will pass through（1）Treated polypide is taken out, and under the conditions of 4 DEG C, distilled water is rinsed, then at successively using concentration at room temperature
The ethanol solution that gradient is 70%, 80%, 90%, 95%, 100% is dehydrated, and the processing time of each gradient is 20 minutes；Distilled water with
Ethanol consumption and the volume ratio of polypide are 50:1；
（3）Transparency of organization：
It will pass through（2）Treated, and polypide is taken out, at room temperature successively in dimethylbenzene：Ethyl alcohol 1:3 mixed liquors, dimethylbenzene：Ethyl alcohol 1:1
It impregnates 10 minutes in mixed liquor, then is impregnated 5 minutes in pure xylene solution；
（4）Waxdip, embedding and slice；
It will pass through（3）Treated, and polypide is taken out, in 55 DEG C of atoleine：Dimethylbenzene 1:It is impregnated 2 hours in 1 mixed liquor, then
It is impregnated 4 hours in 60 DEG C of atoleine, takes out, be sliced after supercooling, solidification, setting；
（5）It opens up piece and dries piece：
It takes slice to move to smear bonding die agent and be preheated on 55 DEG C of glass slide, slice is unfolded completely naturally immediately, then is placed in
12 hours baking pieces under the conditions of 37 DEG C；The group of bonding die agent becomes：Protein liquid, glycerol, sodium salicylate；Its ratio be 10:10:1；
（6）Dewaxing and tissue rehydration：
Take dry piece after glass slide be immersed in xylene solution, keep 5 minutes, then successively soaking concentration gradient be 100%,
95%, 90%, 80%, 70% ethanol solution, then be soaked in distilled water, the processing time of each gradient is 1 ~ 3 minute；
（7）Dyeing：
Glass slide after taking tissue rehydration is immersed in hematoxylin solution, is kept for 5 minutes, is moved in distilled water and rinse 1 minute, then
It moves to and returns blue liquid immersion 1 minute, 30 seconds in differentiation liquid, finally dyed 2 minutes in the solution of Yihong；
（8）Dehydration with it is transparent：
Take dyeing after glass slide carry out distilled water rinse 5 minutes, then be successively immersed in concentration gradient be 70%, 80%, 90%,
95%, it 2 ~ 5 minutes in 100% ethanol solution, moves in xylene solution and impregnates 3 minutes；
（9）Mounting and observation
Neutral gum is added dropwise on biopsy tissues, covered is placed in draught cupboard after being dried, and is seen under the microscope
It examines.
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| CN201610226169.8A CN105928752B (en) | 2016-04-13 | 2016-04-13 | A kind of insect imago paraffin section colouring method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107102017B (en) * | 2017-05-16 | 2019-11-08 | 西北农林科技大学 | The method that same sample is observed using paraffin section and scanning electron microscope |
| CN109141997A (en) * | 2018-08-03 | 2019-01-04 | 福建农林大学 | A kind of flaking method of microexamination Aphytis |
| CN110441117A (en) * | 2019-08-05 | 2019-11-12 | 安徽省农业科学院水产研究所 | A kind of Quantitative Western silver staining of freshwater ciliate |
| CN110672395A (en) * | 2019-11-19 | 2020-01-10 | 李雄 | HE staining method for tissue section |
| CN114839025B (en) * | 2022-04-15 | 2024-04-12 | 中南大学 | Cadaver fly pupae tissue paraffin section staining method |
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| CN102388860A (en) * | 2011-10-18 | 2012-03-28 | 沈阳大学 | Method for preparing small forensic insect specimens |
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| CN105181406A (en) * | 2015-08-14 | 2015-12-23 | 东北林业大学 | Method for making paraffin section of elytrum of insect |
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| CN102388860A (en) * | 2011-10-18 | 2012-03-28 | 沈阳大学 | Method for preparing small forensic insect specimens |
| CN103759994A (en) * | 2014-01-02 | 2014-04-30 | 河南科技大学 | Larva paraffin section forming method |
| CN105181406A (en) * | 2015-08-14 | 2015-12-23 | 东北林业大学 | Method for making paraffin section of elytrum of insect |
| CN105466746A (en) * | 2016-01-21 | 2016-04-06 | 河南科技大学 | Paraffin section adhibiting agent formula and preparation method thereof |
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