CN105928752A - Method for staining paraffin section of adult insect - Google Patents
Method for staining paraffin section of adult insect Download PDFInfo
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- CN105928752A CN105928752A CN201610226169.8A CN201610226169A CN105928752A CN 105928752 A CN105928752 A CN 105928752A CN 201610226169 A CN201610226169 A CN 201610226169A CN 105928752 A CN105928752 A CN 105928752A
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- 238000000034 method Methods 0.000 title claims abstract description 42
- 241000238631 Hexapoda Species 0.000 title claims abstract description 21
- 239000012188 paraffin wax Substances 0.000 title claims abstract description 15
- 238000010186 staining Methods 0.000 title abstract description 5
- 239000007788 liquid Substances 0.000 claims abstract description 11
- 230000018044 dehydration Effects 0.000 claims abstract description 8
- 238000006297 dehydration reaction Methods 0.000 claims abstract description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 36
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 27
- 239000012153 distilled water Substances 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 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
- 238000004043 dyeing Methods 0.000 claims description 7
- 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
- 238000004040 coloring Methods 0.000 claims description 6
- 229940057995 liquid paraffin Drugs 0.000 claims description 6
- 239000000834 fixative Substances 0.000 claims description 5
- 230000008520 organization Effects 0.000 claims description 5
- 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
- 239000003517 fume Substances 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
- 238000004781 supercooling Methods 0.000 claims description 3
- 238000003860 storage Methods 0.000 claims description 2
- 210000000056 organ Anatomy 0.000 abstract description 7
- 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
- 230000007812 deficiency Effects 0.000 abstract description 2
- 210000002249 digestive system Anatomy 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
- 238000013081 phylogenetic analysis Methods 0.000 abstract description 2
- 238000001035 drying Methods 0.000 abstract 1
- 238000007789 sealing Methods 0.000 abstract 1
- 210000001835 viscera Anatomy 0.000 abstract 1
- 239000001993 wax Substances 0.000 abstract 1
- 239000002917 insecticide Substances 0.000 description 4
- 239000012530 fluid Substances 0.000 description 2
- 230000003187 abdominal effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 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)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention provides a method for staining a paraffin section of adult insect, and belongs to the technical field of microscopic tissue section. The method includes tissue fixation, tissue dehydration, tissue transparency, waxdip, embedding and sectioning, section-flatting and section-drying, lost-wax and tissue rehydration, staining, dehydration and transparency, and section-sealing and observation. The final section has high distinction degree of the tissue, and the space structure of each organ and tissue of the insect body can be observed clearly. The method is used for the adult insect body material with hard body wall and low content of the body cavity liquid. The internal digestive system, excretory system, nervous system, respiratory system, circulatory system, and reproductive system can be observed clearly under the microscope by using the method to process the phorid adult insect body. The method for overall paraffin section of adult insect can fill the gap of observation of adult insect internal organ and tissue, can make up for the deficiency of partial observation, can provide classification feature of insect phylogenetic analysis, and has simple operation, short time consuming, and low cost.
Description
Technical field
The present invention relates to a kind of insect imago paraffin section colouring method, belong to microscopic tissue sections technical field, especially
Relate to a kind of insect imago paraffin section colouring method.
Background technology
Insecticide paraffin section and staining technique are research insect tissue form and the important experimental technique of histopathology, typically
Including dissect, fix, be dehydrated transparent, embed, cut into slices, the step such as dyeing.Paraffin section and staining technique are used for insecticide at present
Larva and the local organization of adult or organ, such as feeler, digestive tract, foot etc..The most not it is observed that insect imago is internal overall
Organ and the space structure of tissue.Internal's form of adult not only reflects its physiological feature, and due to internal structure than
Formalness is affected by environment little, the most more has the stability planted.But because adult ectoskeleton is hard, cavity fluids content is few,
The most conventional dicing method is difficult to make complete paraffin section；And the dicing method of adult local organization or organ is not only grasped
Make complexity, the longest, and often need the more senior instrument and equipments such as negative pressure of vacuum process, it is often more important that finally fix, embed, cut
The treatment effect of sheet etc. is not ideal enough, and the section of final local is extremely limited to tissue area indexing, it is difficult in observing complete polypide
The each system in portion and the space structure of organ.
Summary of the invention
It is an object of the invention to provide a kind of insect imago paraffin section colouring method, the method is the most simple to operate, consumption
Time short, low cost, additionally it is possible to be clearly observable each organ and the space structure of tissue in polypide.
The technical solution adopted in the present invention is:
A kind of insect imago paraffin section colouring method, comprises the following steps:
(1) tissue is fixed:
Remove except the insect body of foot and wing is dipped in tissue fixative solution, left at room temperature 1 hour, at 4 DEG C, stand 12 little
Time；Tissue fixative solution is: concentration is the TritonX-100-4% paraformaldehyde mixed stationary liquid of 0.5%.Storage temperature is subzero 20
DEG C, consumption and body volume are than for 50:1.
(2) tissue dewatering:
To take out through the polypide after (1) processes, under the conditions of 4 DEG C, distilled water rinses, then uses concentration the most successively
Gradient is the ethanol solution dehydration of 70%, 80%, 90%, 95%, 100%, and the process time of each gradient is 20 minutes；Distilled water with
Ethanol consumption is 50:1 with the volume ratio of polypide.
(3) transparency of organization:
To take out through the polypide after (2) process, successively at dimethylbenzene under room temperature: ethanol 1:3 mixed liquor, dimethylbenzene: ethanol 1:1
Mixed liquor soaks 10 minutes, then soaks 5 minutes in pure xylene solution；
(4) waxdip, embed and cut into slices；
To take out through the polypide after (3) process, the liquid paraffin at 55 DEG C: dimethylbenzene 1:1 mixed liquor soaks 2 hours, then
In the liquid paraffin of 60 DEG C soak 4 hours, through supercooling, solidify, shape after taking-up, cut into slices；
(5) exhibition sheet and baking sheet:
Taking section move to smear bonding die agent and be preheated on the microscope slide of 55 DEG C, section is unfolded the most completely, then is placed in
Within under the conditions of 37 DEG C 12 hours, dry sheet；Consisting of of bonding die agent: protein liquid, glycerol, sodium salicylate.Its ratio is 10:10:1.
(6) dewax and organize rehydration:
Take and dry the microscope slide after sheet and be immersed in xylene solution, keep 5 minutes, more successively soaking concentration gradient be 100%,
95%, the ethanol solution of 90%, 80%, 70%, then be soaked in distilled water, the process time of each gradient is 1 ~ 3 minute.
(7) dyeing:
Take the microscope slide after tissue rehydration to be immersed in hematoxylin solution, keep 5 minutes, move to distilled water rinses 1 minute, then
Move to return blue immersion steep 1 minute, in differentiation liquid 30 seconds, finally dye 2 minutes in the solution of Yihong.
(8) dehydration is with transparent:
Take the microscope slide after dyeing to carry out distilled water and rinse 5 minutes, then be immersed in successively Concentraton gradient be 70%, 80%, 90%,
95%, in the ethanol solution of 100% 2 ~ 5 minutes, move to xylene solution soaks 3 minutes.
(9) mounting and observation
Biopsy tissues drips neutral gum, covered, is placed in after fume hood dries, sees under the microscope
Examine.
Beneficial effects of the present invention:
Insect imago paraffin section method in the present invention, simple to operate, the shortest, low cost, tissue area is indexed by final section
High, it is possible to be clearly observable each organ and the space structure of tissue in polypide, it is adaptable to body wall is hard, cavity fluids content relatively
Few adult polypide material.Use the method to process eye fly adult polypide, inside can be clearly observed under the microscope and disappear
Change system, Excretory system, nervous system, respiratory system, blood circulation and reproductive system.Insect imago entirety paraffin of the present invention is cut
Sheet method is possible not only to fill up the blank of insect imago internal and structure observation, it is also possible to make up the deficiency that local is observed,
Characteristic of division is provided for insecticide Phylogenetic Analysis.
Accompanying drawing explanation
Fig. 1 is female eye fly adult abdominal part sectional view；
Fig. 2 is male eye fly adult entirety rip cutting figure.
Detailed description of the invention
The present invention is only described in further detail by following embodiment, but does not constitute any limitation of the invention.
Embodiment 1
The present embodiment is eye fly Paraffin Sections of Adult Worm method, comprises the following steps:
1, tissue is fixing
The TritonX-100-4% paraformaldehyde mixed stationary liquid of compound concentration 0.5% in chemical hood.
Fixing flow process is: examination is collected for polypide and is placed on sled, directly renders to mixing fixing after removing foot and wing
In liquid, the amount of fixative and body volume ratio for 50:1, stands 12 hours at a temperature of 4 DEG C；Test insecticide is artificial Intelligent culture
Case is raised eye fly adult, raising temperature 26 DEG C, relative humidity 70%
2, tissue dewatering
To take out through the polypide after (1) processes, under the conditions of 4 DEG C, distilled water rinses, then uses concentration the most successively
Gradient is the ethanol solution dehydration of 70%, 80%, 90%, 95%, 100%, and the process time of each gradient is 20 minutes；Distilled water with
Ethanol consumption is 50:1 with the volume ratio of polypide.
3, transparency of organization:
To take out through the polypide after (2) process, successively at dimethylbenzene under room temperature: ethanol 1:3 mixed liquor, dimethylbenzene: ethanol 1:1
Mixed liquor soaks 10 minutes, then soaks 5 minutes in pure xylene solution；
4, waxdip, embed and cut into slices；
To take out through the polypide after (3) process, the liquid paraffin at 55 DEG C: dimethylbenzene 1:1 mixed liquor soaks 2 hours, then
In the liquid paraffin of 60 DEG C soak 4 hours, through supercooling, solidify, shape after taking-up, cut into slices；
5, exhibition sheet and baking sheet:
Taking section move to smear bonding die agent and be preheated on the microscope slide of 55 DEG C, section is unfolded the most completely, then is placed in
Within under the conditions of 37 DEG C 12 hours, dry sheet；Consisting of of bonding die agent: protein liquid, glycerol, sodium salicylate.Its ratio is 10:10:1.
6, dewax and organize rehydration:
Take and dry the microscope slide after sheet and be immersed in xylene solution, keep 5 minutes, more successively soaking concentration gradient be 100%,
95%, the ethanol solution of 90%, 80%, 70%, then be soaked in distilled water, the process time of each gradient is 1 ~ 3 minute.
7, dyeing:
Take the microscope slide after tissue rehydration to be immersed in hematoxylin solution, keep 5 minutes, move to distilled water rinses 1 minute, then
Move to return blue immersion steep 1 minute, in differentiation liquid 30 seconds, finally dye 2 minutes in the solution of Yihong.
8, dehydration is with transparent:
Take the microscope slide after dyeing to carry out distilled water and rinse 5 minutes, then be immersed in successively Concentraton gradient be 70%, 80%, 90%,
95%, in the ethanol solution of 100% 2 ~ 5 minutes, move to xylene solution soaks 3 minutes.
9, mounting and observation
Biopsy tissues drips neutral gum, covered, is placed in after fume hood dries, sees under the microscope
Examine.
Internal digestive system, Excretory system, nervous system, respiratory system can be clearly observed under the microscope, follow
Loop systems and reproductive system are shown in Fig. 1, Fig. 2.
Claims (1)
1. an insect imago paraffin section colouring method, it is characterised in that described method includes procedure below:
Tissue is fixing:
Remove except the insect body of foot and wing is dipped in tissue fixative solution, left at room temperature 1 hour, at 4 DEG C, stand 12 little
Time；Tissue fixative solution is: concentration is the TritonX-100-4% paraformaldehyde mixed stationary liquid of 0.5%；Storage temperature is subzero 20
DEG C, consumption and body volume are than for 50:1；
(2) tissue dewatering:
To take out through the polypide after (1) processes, under the conditions of 4 DEG C, distilled water rinses, then uses concentration the most successively
Gradient is the ethanol solution dehydration of 70%, 80%, 90%, 95%, 100%, and the process time of each gradient is 20 minutes；Distilled water with
Ethanol consumption is 50:1 with the volume ratio of polypide；
(3) transparency of organization:
To take out through the polypide after (2) process, successively at dimethylbenzene under room temperature: ethanol 1:3 mixed liquor, dimethylbenzene: ethanol 1:1
Mixed liquor soaks 10 minutes, then soaks 5 minutes in pure xylene solution；
(4) waxdip, embed and cut into slices；
To take out through the polypide after (3) process, the liquid paraffin at 55 DEG C: dimethylbenzene 1:1 mixed liquor soaks 2 hours, then
In the liquid paraffin of 60 DEG C soak 4 hours, through supercooling, solidify, shape after taking-up, cut into slices；
(5) exhibition sheet and baking sheet:
Taking section move to smear bonding die agent and be preheated on the microscope slide of 55 DEG C, section is unfolded the most completely, then is placed in
Within under the conditions of 37 DEG C 12 hours, dry sheet；Consisting of of bonding die agent: protein liquid, glycerol, sodium salicylate；Its ratio is 10:10:1；
(6) dewax and organize rehydration:
Take and dry the microscope slide after sheet and be immersed in xylene solution, keep 5 minutes, more successively soaking concentration gradient be 100%,
95%, the ethanol solution of 90%, 80%, 70%, then be soaked in distilled water, the process time of each gradient is 1 ~ 3 minute；
(7) dyeing:
Take the microscope slide after tissue rehydration to be immersed in hematoxylin solution, keep 5 minutes, move to distilled water rinses 1 minute, then
Move to return blue immersion steep 1 minute, in differentiation liquid 30 seconds, finally dye 2 minutes in the solution of Yihong；
(8) dehydration is with transparent:
Take the microscope slide after dyeing to carry out distilled water and rinse 5 minutes, then be immersed in successively Concentraton gradient be 70%, 80%, 90%,
95%, in the ethanol solution of 100% 2 ~ 5 minutes, move to xylene solution soaks 3 minutes；
(9) mounting and observation
Biopsy tissues drips neutral gum, covered, is placed in after fume hood dries, sees under the microscope
Examine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610226169.8A CN105928752B (en) | 2016-04-13 | 2016-04-13 | A kind of insect imago paraffin section colouring method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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 | Publication Date |
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| CN105928752A true CN105928752A (en) | 2016-09-07 |
| CN105928752B CN105928752B (en) | 2018-11-20 |
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| CN201610226169.8A Expired - Fee Related CN105928752B (en) | 2016-04-13 | 2016-04-13 | A kind of insect imago paraffin section colouring method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107102017A (en) * | 2017-05-16 | 2017-08-29 | 西北农林科技大学 | The method observed using paraffin section and ESEM same sample |
| 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 |
| CN114839025A (en) * | 2022-04-15 | 2022-08-02 | 中南大学 | Paraffin section staining method for pupa tissue of autophagous flies |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107102017A (en) * | 2017-05-16 | 2017-08-29 | 西北农林科技大学 | The method observed using paraffin section and ESEM same sample |
| 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 |
| CN114839025A (en) * | 2022-04-15 | 2022-08-02 | 中南大学 | Paraffin section staining method for pupa tissue of autophagous flies |
| CN114839025B (en) * | 2022-04-15 | 2024-04-12 | 中南大学 | Cadaver fly pupae tissue paraffin section staining method |
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| CN105928752B (en) | 2018-11-20 |
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Granted publication date: 20181120 Termination date: 20190413 |