CN103115809A - Transmission electron microscope processing method for insect antenna samples - Google Patents
Transmission electron microscope processing method for insect antenna samples Download PDFInfo
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- CN103115809A CN103115809A CN2013100261562A CN201310026156A CN103115809A CN 103115809 A CN103115809 A CN 103115809A CN 2013100261562 A CN2013100261562 A CN 2013100261562A CN 201310026156 A CN201310026156 A CN 201310026156A CN 103115809 A CN103115809 A CN 103115809A
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Abstract
The invention belongs to the field of experimental sample processing technologies, relates to insect antenna sample processing methods and particularly relates to a transmission electron microscope processing method for insect antenna samples. The method sequentially comprises the following steps of: (A) preparing fixing liquid; (B) preparing various embedding agents; (C) dissecting, fixing and rinsing; (D) dewatering and soaking; and (E) gathering, so as to obtain the samples. The method has the advantages that the problems of difficulty in fixing liquid soaking and insufficiency in embedding agent soaking during the process of insect antenna transmission processing are solved, finally-obtained sample slices can be relatively flat, the phenomena of sample wrinkling, damaging and losing are greatly reduced, internal structures of the sample slices are all effectively fixed, and subcellular structures are clearly visible.
Description
Technical field
The invention belongs to the laboratory sample processing technology field, relate to a kind of sample treatment of insect feeler, relate in particular to a kind of sample for use in transmitted electron microscope disposal route of insect feeler.
Background technology
The commercialization transmission electron microscope, since the 1980's ends generally came into the market, has been brought into play very big effect at life science.Conventional TEM sample preparation technology is also very ripe.Yet, at the biological sample that research organization is fine and close, ossification intensity is higher---during such as the insect feeler, still there is certain technical bottleneck.
Being distributed with a large amount of perceptrons on the feeler of insect, is the very important sense organ of insect, finds spouse and host plant insect, hides in the important ecological behavior such as harmful animal and play key effect.The ultrastructure of research insect feeler, for significant from more profound sensing mechanism and the ecological behavior of understanding insect.Therefore, in recent years, utilize the Ultrastructural report of transmission electron microscopy research insect feeler increasingly extensive.
The feeler epidermis of insect is the part of its body wall.The body wall of insect is its outermost layer tissue; by single cell layer and secretion thereof, formed, comprising fine and close wax layer, protect the wax layer, through tanning contain SPP1 layer etc., be the protective barrier of insect; the evaporation of moisture in insect bodies can be prevented, the invasion of foreign matter can be prevented again.Therefore, insect feeler case-hardening degree is higher, dense structure, when it is carried out to conventional transmission electron microscopy sample preparation, immobile liquid and embedding medium often are difficult to fully soak into sample tissue, thereby make the feeler inner structure can not get fast and effeciently fixing, in the transmission sample section, produce many white spaces; And embedding medium can not fully soak into sample and makes resin and sample easily throw off, misplace, produce gap when the embedded block polymerization, thereby make sample drag in section that in sheet, dyeing, rinse cycle, easily fold, breakage or even monoblock are lost.In addition, imbedded mold by embedding plate as the Spon resin, be beneficial to very much the whole appearance operation of insect feeler, be conducive to the section work of subsequent sample, but because the Spon resin viscosity is larger, when the insect feeler higher in order to the embedding ossification intensity, more be difficult for soaking into, the fragile position of insect feeler (as the flagellum of thorn aristiform feeler) is very easy to lose in tough embedding medium simultaneously.And the Spurr mobility is better, be more suitable for embedding insect feeler, but the Spurr resin is bitten oxygen, commonly use the 0.5ml centrifuge tube in normal experiment as its imbedded mold, and whole appearance operation extremely difficult carry out of insect feeler in the 0.5ml centrifuge tube.In addition, the fixing agent rinsing is unclean, causes osmium calculation and aldehyde fixative or alcohols dehydration agent generation redox reaction, produces precipitation, affects the attractive in appearance of sample section and observes.
Fixing and soak into difficulty ubiquitous technical matters while being the higher biological sample of application transmission electron microscopy research compact tissue and ossification intensity, this is particularly outstanding when research insect feeler ultrastructure.When the insect feeler being carried out to the transmission processing, also there are some problems in the choice and operation of embedding medium.Therefore, we improve conventional transmission sample making technology, so that insect feeler ultrastructure is presented better, have solved the problem that Spon and Spurr embedding medium occur when embedding insect feeler.Simultaneously also to solving the Similar Problems that this technology occurs when being applied to other research fields, provide certain reference.
Summary of the invention
The present invention is directed to defect of the prior art, purpose is to provide a kind of sample for use in transmitted electron microscope disposal route of insect feeler, by using novel immobile liquid, low temperature to dissect, vacuumize that front cushion sample immerses immobile liquid and low temperature manually vacuumizes, make the inner ultrastructure of insect feeler obtain effectively fixing and preserve.By using New Buffering liquid and being decoloured at decolorization swinging table, make the rinsing of fixing agent more thorough.Soak into and adopt soak process to carry out at decolorization swinging table by gradient, making embedding medium more fully infiltrate sample, especially having solved the problem that the larger Spon resin of viscosity is difficult for soaking into sample; By preparing novel embedding medium, make embedding medium hardness and sample hardness more approaching, make embedding medium and sample polymerization good.In addition, to rinsing and low-concentration ethanol solution dehydrates after front fixing, also adopt low temperature to carry out, fixing, rinsing, dehydration, soak process are all carried out at decolorization swinging table, have reduced as possible degraded and the distortion of sample composition in processing procedure, are conducive to cyto-architectural preservation.Adopt embedding plate as imbedded mold, be more conducive to whole appearance and the subsequent slice of sample, the Spurr polymerization the time encapsulation process solved the problem that the Spurr resin is bitten oxygen.The invention solves in the insect feeler is carried out to the sample for use in transmitted electron microscope processing procedure, immobile liquid soak into difficulty, embedding medium soak into insufficient, the fixing agent rinsing is clean, the problem of the generation of the whole appearance difficulty of sample, the inconvenient subsequent slice of sample embedded block and dyeing precipitation.
In order to solve the problems of the technologies described above, the present invention is solved by following technical proposals:
A kind of sample for use in transmitted electron microscope disposal route of insect feeler, be comprised of following step successively,
A. the preparation of immobile liquid: in the PBS damping fluid containing paraformaldehyde and glutaraldehyde, add sucrose, 4 ℃ of preservations, wherein the PBS damping fluid is phosphate buffer;
B. the preparation of various embedding mediums:
(1) SPI-Pon
tMthe preparation of 812 embedding mediums: by SPI – Pon812, DDSA, NMA and DMP – 30, by sample, add in beaker, with magnetic stirring apparatus, stir evenly, after four kinds of compositions add entirely, with aluminium-foil paper sealed beaker mouth, continue to stir approximately 1 hour, remove aluminium-foil paper, with vacuum dryer, the bubble in embedding medium is extracted out, and in exsiccator standing a moment treat that bubble disappears fully, the embedding medium prepared is transferred in the syringe of dry centrifuge tube or sealing, be sealed in the polybag that is placed with silica-gel desiccant, 20 ℃ of preservations of –, during use, take out in advance, room temperature to be returned to can be used, wherein SPI – Pon812 is epoxy resin, DDSA is 2-dodecenylsuccinic acid acid anhydride, NMA is the methyl carbic anhydride, DMP-30 is 2-4-6-tri-(dimethylamino methyl) phenol,
(2) preparation of Spurr (4221) embedding medium: by ERL 4221, DER 736, NSA and DMAE, by sample, add in beaker, with magnetic stirring apparatus, stir evenly, after four kinds of compositions add entirely, with aluminium-foil paper sealed beaker mouth, continue to stir approximately 1 hour, remove aluminium-foil paper, with vacuum dryer, the bubble in embedding medium is extracted out, and in exsiccator standing a moment treat that bubble disappears fully, the embedding medium prepared is transferred in the syringe of dry centrifuge tube or sealing, be sealed in the polybag that is placed with silica-gel desiccant, 20 ℃ of preservations of –, during use, take out in advance, room temperature to be returned to can be used, wherein ERL 4221 is epoxy resin, DER 736 is polyglycol type epoxy resins, NSA is the nonenyl succinic acid acid anhydride, DMAE is dimethyl ethanolamine,
C. dissect, fix and rinsing: get insect imago, put into the bottle that fills immobile liquid, in immobile liquid, insect imago is killed, insect head under dissecting in immobile liquid, put into another bottle that fills immobile liquid, the bottle seal that will fill sample with the rubber peel plug is good, manually sample is carried out to vacuum pumping with syringe, until sample sinks fully, sample in bottle is transferred in the centrifuge tube that fresh immobile liquid is housed, front fixing, front rinsing, carry out rear fixing, post rinse with osmic acid solution again;
D. dewater and soak into: carry out processed with gradient ethanolic solution and the epoxypropane sample good to rinsing, then with the mixed liquor of epoxypropane and embedding medium, sample is carried out to gradient and soak into, then replace tubes, soak into sample with embedding medium;
E. polymerization: by the exhaustion of the resin of sample surfaces, put on embedding plate the embedding hole that fills embedding medium, then carry out whole appearance processing, embedding plate is put into to the baking oven heating and make the embedding medium polymerization, sample preparation completes.
As preferably, the preparation of the immobile liquid in steps A, containing the 2%(massfraction) paraformaldehyde and 2.5%(massfraction) in the 0.1mol/L PBS damping fluid (pH7.4) of glutaraldehyde, add the 5%(massfraction) sucrose, 4 ℃ of preservations, wherein phosphate buffer is kaliumphosphate buffer or sodium phosphate buffer.
As preferably, the preparation of the immobile liquid in steps A, add the 5%(massfraction) after sucrose, also to add 1% tannic acid, 4 ℃ of preservations.
As preferably, the preparation of (1) SPI-PonTM 812 embedding mediums in step B: SPI – Pon812, DDSA, NMA and DMP – 30 add respectively 10 parts, 2.2 parts, 8 parts and 0.3 part by weight.
As preferably, the preparation of (2) Spurr (4221) embedding medium in step B: ERL 4221, DER 736, NSA and DMAE add respectively 5 parts, 2.7 parts, 12 parts and 0.18 part by weight.
As preferably, in step C, insect head under dissecting under stereomicroscope, insert head with dissecting needle, make head immerse immobile liquid fully, dissect complete, to fill the bottle seal of sample with the rubber peel plug, all disscting instruments all need to meet cold, and the dissection process keeps the immobile liquid sealing in bottle, reduces its volatilization.
As preferably, syringe in step C comprises syringe needle and the 50ml injector syringe that pin number * needle tubing length is 6 * 30, need frequently slowly to open rubber stopper in vacuum, and shake gently bottle, the sample of ejection is got back in immobile liquid, continue to vacuumize, until sample sinks fully.
As preferably, in step C, sample in bottle is transferred in the centrifuge tube that immobile liquid is housed, be placed on decolorization swinging table fixingly, fix 2～4 hours before under 0～4 ℃ of condition, with rinsing before phosphate buffer 1～10 time, each 10～20min, fix after using again osmic acid solution, then use the phosphate buffer post rinse 1～10 time, each 10～20min.
As preferably, in step C, to dissect and vacuumize on ice and carry out, front fixing and front rinsing is carried out at 0～4 ℃ of chromatography cabinet, and rear fixing and post rinse carries out in room temperature, and front fixing, front rinsing, rear fixing and post rinse all carry out at decolorization swinging table.
As preferably, in step D, use successively 50%, 70%, 80%, 90% and 95% ethanolic solution carries out processed to sample, every kind of concentration is processed 10～20min, use again Ethanol Treatment 15～25min of 99.9%, with epoxypropane, sample is carried out to transition processing 3 times, each 5～15min, by volume ratio, be 3:1 again, 2:1, 1:1, the epoxypropane of 1:2 and 1:3 and the mixed liquor of embedding medium carry out gradient to sample and soak into, processing sample 30min successively, 45min, 1h, 1.5h and 3h, then replace tubes, with embedding medium processing sample 24h, ethanolic solution with 50% and 70% carries out at 0～4 ℃ of chromatography cabinet while dewatering, with 80%, 90%, 95% and 99.9% ethanolic solution all carries out in room temperature while processing, dehydration and soak process are all carried out at decolorization swinging table.
The invention discloses a kind of sample for use in transmitted electron microscope disposal route of insect feeler, by using novel immobile liquid, low temperature to dissect, vacuumize that front cushion sample immerses immobile liquid and low temperature manually vacuumizes, make the inner ultrastructure of insect feeler obtain effectively fixing and preserve.By using New Buffering liquid, make the rinsing of aldehyde fixative more thorough.Soak into and adopt soak process to carry out at decolorization swinging table by gradient, making embedding medium more fully infiltrate sample, especially having solved the problem that the larger Spon resin of viscosity is difficult for soaking into sample; By preparing novel embedding medium, make embedding medium hardness and sample hardness more approaching, make embedding medium and sample polymerization good.Solved in the insect feeler is carried out to the transmission processing procedure, immobile liquid soaks into difficulty, embedding medium soaks into inadequate problem.
In addition, to rinsing and low-concentration ethanol solution dehydrates after front fixing, also adopt low temperature to carry out, fixing, rinsing, dehydration, soak process are all carried out at decolorization swinging table, have reduced as possible degraded and the distortion of sample composition in processing procedure, are conducive to cyto-architectural preservation.By using New Buffering liquid and being decoloured at decolorization swinging table, make the rinsing of fixing agent more thoroughly, reduced the generation of dyeing precipitation.Adopt embedding plate as imbedded mold, be more conducive to whole appearance and the subsequent slice of sample, the encapsulation process during Spurr polymerization has solved the problem that the Spurr resin is bitten oxygen.The sample for use in transmitted electron microscope disposal route of a kind of insect feeler of the present invention, can make the sample finally obtained cut into slices more smooth, sample fold, breakage, Loss greatly reduce, sample section inner structure is all effectively fixed, subcellular structure is clear to be showed, and in front immobile liquid, adds the insect feeler sample cell film profile of tannic acid clear especially.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment 1
The preparation of immobile liquid: in the PBS damping fluid containing paraformaldehyde and glutaraldehyde, add sucrose, 4 ℃ of preservations, wherein the PBS damping fluid is phosphate buffer;
Containing the 2%(massfraction) paraformaldehyde and 2.5%(massfraction) in the 0.1mol/L PBS damping fluid (pH 7.4) of glutaraldehyde, add the 5%(massfraction) sucrose, 4 ℃ of preservations, wherein phosphate buffer is kaliumphosphate buffer or sodium phosphate buffer.
Concrete proportioning is as follows: 0.2mol/L PBS damping fluid (pH 7.4) 50ml, 25%(massfraction) glutaraldehyde 10ml, 10%(massfraction) paraformaldehyde 20ml, the ultrapure water 20ml prepared by the pure water instrument and sucrose 5g, make through being uniformly mixed, and preserve under 4 ℃ of conditions.
Embodiment 2
The preparation of immobile liquid: in the PBS damping fluid containing paraformaldehyde and glutaraldehyde, add sucrose, 4 ℃ of preservations, wherein the PBS damping fluid is phosphate buffer;
Containing the 2%(massfraction) paraformaldehyde and 2.5%(massfraction) in the 0.1mol/L PBS damping fluid (pH 7.4) of glutaraldehyde, add the 5%(massfraction) sucrose, 4 ℃ of preservations, wherein phosphate buffer is kaliumphosphate buffer or sodium phosphate buffer.
Add the 5%(massfraction) after sucrose, also to add 1% tannic acid, 4 ℃ of preservations.
Concrete proportioning is as follows: 0.2mol/L PBS damping fluid (pH 7.4) 50ml, 25%(massfraction) glutaraldehyde 10ml, 10%(massfraction) paraformaldehyde 20ml, the ultrapure water 20ml prepared by the pure water instrument, sucrose 5g and tannic acid 1g, make through being uniformly mixed, and preserve under 4 ℃ of conditions.
Embodiment 3
A kind of sample for use in transmitted electron microscope disposal route of insect feeler, be comprised of following step successively,
A. the preparation of immobile liquid: in the PBS damping fluid containing paraformaldehyde and glutaraldehyde, add sucrose, 4 ℃ of preservations, wherein the PBS damping fluid is phosphate buffer;
B. the preparation of various embedding mediums:
(1) SPI-Pon
tMthe preparation of 812 embedding mediums: by SPI – Pon812, DDSA, NMA and DMP – 30, by sample, add in beaker, with magnetic stirring apparatus, stir evenly, after four kinds of compositions add entirely, with aluminium-foil paper sealed beaker mouth, continue to stir approximately 1 hour, remove aluminium-foil paper, with vacuum dryer, the bubble in embedding medium is extracted out, and in exsiccator standing a moment treat that bubble disappears fully, the embedding medium prepared is transferred in the syringe of dry centrifuge tube or sealing, be sealed in the polybag that is placed with silica-gel desiccant, 20 ℃ of preservations of –, during use, take out in advance, room temperature to be returned to can be used, wherein SPI – Pon812 is epoxy resin, DDSA is 2-dodecenylsuccinic acid acid anhydride, NMA is the methyl carbic anhydride, DMP-30 is 2-4-6-tri-(dimethylamino methyl) phenol,
(2) preparation of Spurr (4221) embedding medium: by ERL 4221, DER 736, NSA and DMAE, by sample, add in beaker, with magnetic stirring apparatus, stir evenly, after four kinds of compositions add entirely, with aluminium-foil paper sealed beaker mouth, continue to stir approximately 1 hour, remove aluminium-foil paper, with vacuum dryer, the bubble in embedding medium is extracted out, and in exsiccator standing a moment treat that bubble disappears fully, the embedding medium prepared is transferred in the syringe of dry centrifuge tube or sealing, be sealed in the polybag that is placed with silica-gel desiccant, 20 ℃ of preservations of –, during use, take out in advance, room temperature to be returned to can be used, wherein ERL 4221 is epoxy resin, DER 736 is polyglycol type epoxy resins, NSA is the nonenyl succinic acid acid anhydride, DMAE is dimethyl ethanolamine,
C. dissect, fix and rinsing: get insect imago, put into the bottle that fills immobile liquid, in immobile liquid, insect imago is killed, insect head under dissecting in immobile liquid, put into another bottle that fills immobile liquid, the bottle seal that will fill sample with the rubber peel plug is good, manually sample is carried out to vacuum pumping with syringe, until sample sinks fully, sample in bottle is transferred in the centrifuge tube that fresh immobile liquid is housed, front fixing, front rinsing, carry out rear fixing, post rinse with osmic acid solution again;
D. dewater and soak into: carry out processed with gradient ethanolic solution and the epoxypropane sample good to rinsing, then with the mixed liquor of epoxypropane and embedding medium, sample is carried out to gradient and soak into, then replace tubes, soak into sample with embedding medium;
E. polymerization: by the exhaustion of the resin of sample surfaces, put on embedding plate the embedding hole that fills embedding medium, then carry out whole appearance processing, embedding plate is put into to the baking oven heating and make the embedding medium polymerization, sample preparation completes.
The preparation of the immobile liquid in steps A, containing the 2%(massfraction) paraformaldehyde and 2.5%(massfraction) in the 0.1mol/L PBS damping fluid (pH 7.4) of glutaraldehyde, add the 5%(massfraction) sucrose, 4 ℃ of preservations, wherein phosphate buffer is kaliumphosphate buffer or sodium phosphate buffer.
The preparation of the immobile liquid in steps A, add the 5%(massfraction) after sucrose, also to add 1% tannic acid, 4 ℃ of preservations.
The preparation of (1) SPI-PonTM 812 embedding mediums in step B: SPI – Pon812, DDSA, NMA and DMP – 30 add respectively 10 parts, 2.2 parts, 8 parts and 0.3 part by weight.
The preparation of (2) Spurr (4221) embedding medium in step B: ERL 4221, DER 736, NSA and DMAE add respectively 5 parts, 2.7 parts, 12 parts and 0.18 part by weight.
In step C, in step C, insect head under dissecting under stereomicroscope, insert head with dissecting needle, make head immerse immobile liquid fully, dissect complete, to fill the bottle seal of sample with the rubber peel plug, all disscting instruments all need to meet cold, and the dissection process keeps the immobile liquid sealing in bottle, reduces its volatilization.
Syringe in step C comprises syringe needle and the 50ml injector syringe that pin number * needle tubing length is 6 * 30, need frequently slowly to open rubber stopper in vacuum, and shake gently bottle, the sample of ejection is got back in immobile liquid, continue to vacuumize, until sample sinks fully.
In step C, sample in bottle is transferred in the centrifuge tube that immobile liquid is housed, be placed on decolorization swinging table fixing, fix 2～4 hours before under 0～4 ℃ of condition, with rinsing before phosphate buffer 1～10 time, each 10～20min, then with fixing after osmic acid solution, use again the phosphate buffer post rinse 1～10 time, each 10～20min.
In step C, dissect and vacuumize on ice and carry out, front fixing and front rinsing is carried out at 0～4 ℃ of chromatography cabinet, and rear fixing and post rinse carries out in room temperature, and front fixing, front rinsing, rear fixing and post rinse all carry out at decolorization swinging table.
In step D, use successively 50%, 70%, 80%, 90% and 95% ethanolic solution carries out processed to sample, every kind of concentration is processed 10～20min, use again Ethanol Treatment 15～25min of 99.9%, with epoxypropane, sample is carried out to transition processing 3 times, each 5～15min, by volume ratio, be 3:1 again, 2:1, 1:1, the epoxypropane of 1:2 and 1:3 and the mixed liquor of embedding medium carry out gradient to sample and soak into, processing sample 30min successively, 45min, 1h, 1.5h and 3h, then replace tubes, with embedding medium processing sample 24h, ethanolic solution with 50% and 70% carries out at 0～4 ℃ of chromatography cabinet while dewatering, with 80%, 90%, 95% and 99.9% ethanolic solution all carries out in room temperature while processing, dehydration and soak process are all carried out at decolorization swinging table.
Concrete operations are as follows: by ready sample, be transferred in the 5ml centrifuge tube that fills fresh immobile liquid, be placed on centrifuge tube shelf, put on the decolorization swinging table that 0～4 ℃ of chromatography cabinet low speed swings up and down, low temperature is 3h fixedly.Outwell immobile liquid, with containing 0.1M PBS (pH7.4) the rinsing sample of 5 ﹪ (massfraction) sucrose three times, each 15min, in 0～4 ℃ of chromatography cabinet, decolorization swinging table carries out, and wherein 0.1M PBS refers to the phosphate buffer that 0.1mol/L pH value is 7.4.Sample is transferred to room temperature, in fuming cupboard with 0.2MPBS (pH7.4) by the 2%(massfraction) the osmic acid solution dilution is 1%, and with 1% osmic acid solution fixed sample 2h, room temperature fuming cupboard decolorization swinging table carries out.With 0.1M PBS rinsing sample three times, each 15min, last rinsing needs replace tubes, and room temperature fuming cupboard decolorization swinging table carries out.Ethanolic solution with 50%, 70%, 80%, 90% and 95% carries out processed to sample, and every kind of concentration is processed 15min, then uses 99.9% Ethanol Treatment 20min, finally uses the epoxypropane transition processing 3 times, each 10min.Processed is all carried out at decolorization swinging table, and low-concentration ethanol carries out at 0～4 ℃ of chromatography cabinet while processing, and sample can be in 4 ℃ of preservations of refrigerator in 70% ethanol, and the operations all since 80% Ethanol Treatment are all carried out in room temperature.After dehydration, the epoxypropane that is 3:1,2:1,1:1,1:2 and 1:3 by volume ratio and the mixed liquor of embedding medium carry out gradient to sample and soak into, successively processing sample 30min, 45min, 1h, 1.5h and 3h, then replace tubes, with embedding medium processing sample 24h, all at the room temperature decolorization swinging table, carry out.In the sample preparation process, must note: change the solution action and want fast, especially do not allow sample leave the immersion of solution, can adopt when changing liquid and retain a small amount of liquid, add treating fluid, when committed step is dewatered as epoxypropane, increase and change liquid number of times etc., guarantee the sample dehydration fully.
Embedding medium is placed in to the embedding hole of 20 hole plane embedding plate, with dissecting forceps, the sample soaked into is taken out from embedding medium, first on filter paper, suck the embedding medium that sample surfaces retains, then put on embedding plate the embedding hole that fills embedding medium, the dissecting needle made from No. 1 insect needle under stereomicroscope is adjusted sample, makes to want the position of sections observation in embedding Kongzui apical position.Should be in the position that approaches baking oven as far as possible during whole appearance, after the whole appearance of sample, it is put into to baking oven gently at once, heating makes the embedding medium polymerization.
If use the Spon embedding medium, sample carried out to gradient polymeric, polymerization 12h under 37 ℃ of conditions; Polymerization 12h under 45 ℃ of conditions; Polymerization 24h under 60 ℃ of conditions.If use the Spurr embedding medium, direct polymerization 8～16h under 70 ℃ of conditions, obtain embedded sample.
While using Spurr embedding sample, embedding plate is packed in molecular biology sample test sample presentation bag commonly used, the most of port of good seal, stay gap to vacuumize with syringe from a side, then sealing fully, puts into baking oven.While using Spurr, in advance on embedding plate appropriate location insert deduct sharp half one dissecting needle to prop certain space, prevent after vacuumizing, embedding medium makes whole appearance with testing the sample presentation bag to contact, and good sample position changes.
Sample is located, is cut into slices in ultramicrotome, obtains the ultra-thin section of 70～90nm.Section, through uranium acetate 50% alcohol saturated solution and the lead citrate solution 30min that dyes respectively, after 25min, is observed, is taken pictures in transmission electron microscope, obtains displaing micro picture.
In a word, the foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to the covering scope of patent of the present invention.
Claims (10)
1. the sample for use in transmitted electron microscope disposal route of an insect feeler is characterized in that: by following step, formed successively,
A. the preparation of immobile liquid: in the PBS damping fluid containing paraformaldehyde and glutaraldehyde, add sucrose, 4 ℃ of preservations, wherein the PBS damping fluid is phosphate buffer;
B. the preparation of various embedding mediums:
(1) SPI-Pon
tMthe preparation of 812 embedding mediums: by SPI – Pon812, DDSA, NMA and DMP – 30, by sample, add in beaker, with magnetic stirring apparatus, stir evenly, after four kinds of compositions add entirely, with aluminium-foil paper sealed beaker mouth, continue to stir approximately 1 hour, remove aluminium-foil paper, with vacuum dryer, the bubble in embedding medium is extracted out, and in exsiccator standing a moment treat that bubble disappears fully, the embedding medium prepared is transferred in the syringe of dry centrifuge tube or sealing, be sealed in the polybag that is placed with silica-gel desiccant, 20 ℃ of preservations of –, during use, take out in advance, room temperature to be returned to can be used, wherein SPI – Pon812 is epoxy resin, DDSA is 2-dodecenylsuccinic acid acid anhydride, NMA is the methyl carbic anhydride, DMP-30 is 2-4-6-tri-(dimethylamino methyl) phenol,
(2) preparation of Spurr (4221) embedding medium: by ERL 4221, DER 736, NSA and DMAE, by sample, add in beaker, with magnetic stirring apparatus, stir evenly, after four kinds of compositions add entirely, with aluminium-foil paper sealed beaker mouth, continue to stir approximately 1 hour, remove aluminium-foil paper, with vacuum dryer, the bubble in embedding medium is extracted out, and in exsiccator standing a moment treat that bubble disappears fully, the embedding medium prepared is transferred in the syringe of dry centrifuge tube or sealing, be sealed in the polybag that is placed with silica-gel desiccant, 20 ℃ of preservations of –, during use, take out in advance, room temperature to be returned to can be used, wherein ERL 4221 is epoxy resin, DER 736 is polyglycol type epoxy resins, NSA is the nonenyl succinic acid acid anhydride, DMAE is dimethyl ethanolamine,
C. dissect, fix and rinsing: get insect imago, put into the bottle that fills immobile liquid, in immobile liquid, insect imago is killed, insect head under dissecting in immobile liquid, put into another bottle that fills immobile liquid, the bottle seal that will fill sample with the rubber peel plug is good, manually sample is carried out to vacuum pumping with syringe, until sample sinks fully, sample in bottle is transferred in the centrifuge tube that fresh immobile liquid is housed, front fixing, front rinsing, carry out rear fixing, post rinse with osmic acid solution again;
D. dewater and soak into: carry out processed with gradient ethanolic solution and the epoxypropane sample good to rinsing, then with the mixed liquor of epoxypropane and embedding medium, sample is carried out to gradient and soak into, then replace tubes, soak into sample with embedding medium;
E. polymerization: by the exhaustion of the resin of sample surfaces, put on embedding plate the embedding hole that fills embedding medium, then carry out whole appearance processing, embedding plate is put into to the baking oven heating and make the embedding medium polymerization, sample preparation completes.
2. the sample for use in transmitted electron microscope disposal route of a kind of insect feeler according to claim 1, it is characterized in that: the preparation of the immobile liquid in steps A, containing the 2%(massfraction) paraformaldehyde and 2.5%(massfraction) in the 0.1mol/L PBS damping fluid (pH7.4) of glutaraldehyde, add the 5%(massfraction) sucrose, 4 ℃ of preservations, wherein phosphate buffer is kaliumphosphate buffer or sodium phosphate buffer.
3. the sample for use in transmitted electron microscope disposal route of a kind of insect feeler according to claim 2, it is characterized in that: the preparation of the immobile liquid in steps A adds the 5%(massfraction) after sucrose, also to add 1% tannic acid, 4 ℃ of preservations.
4. the sample for use in transmitted electron microscope disposal route of a kind of insect feeler according to claim 1, it is characterized in that: the preparation of (1) SPI-PonTM 812 embedding mediums in step B: SPI – Pon812, DDSA, NMA and DMP – 30 add respectively 10 parts, 2.2 parts, 8 parts and 0.3 part by weight.
5. the sample for use in transmitted electron microscope disposal route of a kind of insect feeler according to claim 1, it is characterized in that: the preparation of (2) Spurr (4221) embedding medium in step B: ERL 4221, DER 736, NSA and DMAE add respectively 5 parts, 2.7 parts, 12 parts and 0.18 part by weight.
6. the sample for use in transmitted electron microscope disposal route of a kind of insect feeler according to claim 1, it is characterized in that: in step C, insect head under dissecting under stereomicroscope, insert head with dissecting needle, make head immerse immobile liquid fully, dissect complete, to fill the bottle seal of sample with the rubber peel plug, all disscting instruments all need to meet cold, and the dissection process keeps the immobile liquid sealing in bottle, reduces its volatilization.
7. the sample for use in transmitted electron microscope disposal route of a kind of insect feeler according to claim 6, it is characterized in that: the syringe in step C comprises syringe needle and the 50ml injector syringe that pin number * needle tubing length is 6 * 30, need frequently slowly to open rubber stopper in vacuum, and shake gently bottle, the sample of ejection is got back in immobile liquid, continue to vacuumize, until sample sinks fully.
8. the sample for use in transmitted electron microscope disposal route of a kind of insect feeler according to claim 7, it is characterized in that: in step C, sample in bottle is transferred in the centrifuge tube that immobile liquid is housed, be placed on decolorization swinging table fixingly, fix 2～4 hours before under 0～4 ℃ of condition, with rinsing before phosphate buffer 1～10 time, each 10～20min, fix after using again osmic acid solution, then use the phosphate buffer post rinse 1～10 time, each 10～20min.
9. the sample for use in transmitted electron microscope disposal route of a kind of insect feeler according to claim 8, it is characterized in that: in step C, dissect and vacuumize on ice and carry out, before fixing and front rinsing at 0～4 ℃ of chromatography cabinet, carry out, rear fixing and post rinse carries out in room temperature, and front fixing, front rinsing, rear fixing and post rinse all carry out at decolorization swinging table.
10. the sample for use in transmitted electron microscope disposal route of a kind of insect feeler according to claim 1, it is characterized in that: in step D, use successively 50%, 70%, 80%, 90% and 95% ethanolic solution carries out processed to sample, every kind of concentration is processed 10～20min, use again Ethanol Treatment 15～25min of 99.9%, with epoxypropane, sample is carried out to transition processing 3 times, each 5～15min, by volume ratio, be 3:1 again, 2:1, 1:1, the epoxypropane of 1:2 and 1:3 and the mixed liquor of embedding medium carry out gradient to sample and soak into, processing sample 30min successively, 45min, 1h, 1.5h and 3h, then replace tubes, with embedding medium processing sample 24h, ethanolic solution with 50% and 70% carries out at 0～4 ℃ of chromatography cabinet while dewatering, with 80%, 90%, 95% and 99.9% ethanolic solution all carries out in room temperature while processing, dehydration and soak process are all carried out at decolorization swinging table.
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