CN113418766B - Double-fixation embedding sample preparation method after integral immunization - Google Patents
Double-fixation embedding sample preparation method after integral immunization Download PDFInfo
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- CN113418766B CN113418766B CN202110703166.XA CN202110703166A CN113418766B CN 113418766 B CN113418766 B CN 113418766B CN 202110703166 A CN202110703166 A CN 202110703166A CN 113418766 B CN113418766 B CN 113418766B
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- 230000003053 immunization Effects 0.000 title claims abstract description 9
- 238000005464 sample preparation method Methods 0.000 title claims abstract description 7
- 238000002360 preparation method Methods 0.000 claims abstract description 14
- 238000004321 preservation Methods 0.000 claims abstract description 12
- 229910000489 osmium tetroxide Inorganic materials 0.000 claims abstract description 11
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- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 210000002421 cell wall Anatomy 0.000 claims abstract description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 48
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
- 229920002866 paraformaldehyde Polymers 0.000 claims description 10
- 239000003822 epoxy resin Substances 0.000 claims description 8
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/02—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by transmitting the radiation through the material
- G01N23/04—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by transmitting the radiation through the material and forming images of the material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/20—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
- G01N23/20008—Constructional details of analysers, e.g. characterised by X-ray source, detector or optical system; Accessories therefor; Preparing specimens therefor
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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
- G01N1/36—Embedding or analogous mounting of samples
- G01N2001/364—Embedding or analogous mounting of samples using resins, epoxy
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- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention discloses a double-fixation embedding sample preparation method after integral immunization. The method is characterized in that a primary antibody and a secondary antibody are combined with an antigen before conventional sample preparation of a transmission electron microscope sample, and then the transmission electron microscope sample preparation is carried out. The invention firstly combines the primary antibody and the secondary antibody before the conventional sample preparation, so that the problem of activity of the antigen is not needed to be worried, and the problems of poor contact between the antigen and the primary antibody and the secondary antibody are solved by opening a channel on the cell wall and the cell membrane through the educing enzyme and the Triton-100, the success rate of marking is improved, glutaraldehyde and osmium tetroxide can be used in the later steps according to the sample preparation process of the biological tissue block, and the problem that the antigen activity preservation and the tissue structure preservation are difficult to be compatible is solved.
Description
Technical field:
the invention belongs to the field of transmission electron microscopes, and particularly relates to a double-fixation embedding sample preparation method after integral immunization.
The background technology is as follows:
transmission electron microscopy is the microscope with the highest resolution available. The transmission electron microscope can be used for observing the ultrastructure of the tissue, and the expected distribution of enzymes, specific ions and antigens (proteins in most cases) in the organelle can be known by combining enzyme positioning, ion positioning and immune nanogold marking technologies.
Transmission electron microscopy requires that the sample be very thin and free of water, so biological tissue typically needs to undergo an aldehyde fixing-osmium tetroxide fixing-dehydration-embedding-ultra-microtome-heavy metal staining process before it can be viewed in transmission electron microscopy. The conventional immune labeling technology is to combine antigen with the first antibody after the ultrathin section is made and then combine the first antibody with the second antibody containing nano gold particles. The gold particles are imaged as black dots, which are different from the tissue imaging results, so that the distribution of the antigen in the cells can be obtained (the antigen is marked to obtain the positioning information). Since glutaraldehyde and osmium tetroxide in the aldehyde-based fixing solution can completely denature an antigen and cannot be combined with the primary antibody, two fixing agents, namely glutaraldehyde and osmium tetroxide, are often not used in the preparation process of a sample which needs to be subjected to the subsequent immunogold marking, and the activity of the antigen is ensured by sacrificing the fixing effect of a tissue structure. However, immobilization is a very critical step in sample preparation, and aims to terminate the biochemical process of cells, stabilize the cellular material components, maintain the original microstructure of living cells, avoid autolysis of cells and spoilage due to invasion of external microorganisms, create artifacts, and create crosslinks between molecules of some components by chemical or physical reactions to provide a scaffold to stabilize the spatial configuration of various organelles, and the fixative osmium tetroxide can also enhance image contrast effects. Because only paraformaldehyde is used as a fixing agent, the immune marked sample is usually not well fixed, and finally the tissue structure is poor, even antigens are very sensitive and easy to deform, and the tissue structure and the antigen activity can not be well preserved in the whole experimental process.
Disclosure of Invention
The invention aims to provide a double-fixation embedding sample preparation method after integral immunization, wherein the whole immunization is complete in antigen activity preservation and tissue structure preservation.
The invention relates to a double-fixing embedding sample preparation method after integral immunization, which is characterized in that a primary antibody and a secondary antibody are combined with an antigen before conventional sample preparation (aldehyde fixing-osmium tetroxide fixing-dehydration-embedding) of a transmission electron microscope sample, and then the transmission electron microscope sample preparation is carried out.
Preferably, the immobilized material is treated with an eductase and/or Triton-100 prior to binding of the primary antibody to the antigen, such that channels are opened in the cell wall and cell membrane of the material.
Preferably, the specific steps of treating the immobilized material with an eductase and/or Triton-100 prior to binding of the primary antibody to the antigen are: cutting the tissue into blocks, fixing, placing the material washed by the phosphate buffer solution into the enzymolysis solution of the educing enzyme for digestion, then washing, then placing into the solution of Triton X-100 for treatment, and then washing by the phosphate buffer solution.
Further preferably, the eductase is 0.15% eductase by mass, and the Triton X-100 solution is 0.1% -0.2% Triton X-100 solution by mass.
Preferably, the specific steps are as follows:
A. cutting tissues into blocks, fixing the blocks by using paraformaldehyde with the mass fraction of 4%, digesting cell walls by using an enzymolysis liquid of a segregation enzyme with the mass fraction of 0.15%, soaking a sealing material by using skimmed milk powder with the mass fraction of 1%, and then incubating a primary antibody and a secondary antibody successively;
B. the incubated material is fixed by a solution containing 2% of paraformaldehyde and 2.5% of glutaraldehyde in mass fraction and by osmium tetroxide in mass fraction of 1%, and the fixed material is cleaned, dehydrated, transited, permeated, embedded and polymerized to obtain the epoxy resin embedded block.
The epoxy resin embedded block can be observed by a transmission electron microscope after ultrathin section and dyeing.
Further preferably, the specific steps are as follows:
a. fixing for the first time: cutting the tissue into small blocks, wherein the maximum thickness is not more than 500 micrometers, and placing the small blocks in a phosphate buffer solution of paraformaldehyde with the mass fraction of 4% for air extraction and preservation for 2 hours at 4 ℃;
b. cleaning for the first time: washing the fixed material with phosphate buffer solution for 5 times, each time for 5min at 4 ℃;
c. digesting the cell wall: preparing an enzymolysis solution containing 0.15% of isolated enzyme by mass percent by using a phosphate buffer solution, placing the tissue block in the step b into the enzymolysis solution, digesting the material for 45min at room temperature, and then washing for 3 times by using the phosphate buffer solution at 4 ℃ for 5min each time;
d. membrane permeation: placing the material in the step c into a phosphate buffer solution containing 0.1-0.2% of TritonX-100 by mass fraction, treating the material at 4 ℃ for 30min, and then washing the material with the phosphate buffer solution for 5 times each time for 5min;
e. closing: soaking the sealing material for 35min by using 1% of skimmed milk powder dissolved in phosphoric acid buffer solution at 4 ℃;
f. an antibody: incubating the material for 1h with a primary antibody solution dissolved in phosphate buffer at 4 ℃;
g. secondary antibody colloidal gold: washing the material with phosphate buffer solution at 4deg.C for 5min for 3 times; incubating the material for 1h with a secondary antibody colloidal gold solution dissolved in a phosphate buffer, and washing the material with the phosphate buffer for 5 times, each time for 5min;
e. second fixation: re-fixing the material by using a phosphate buffer solution containing 2% of paraformaldehyde and 2.5% of glutaraldehyde by mass, placing the material in a refrigerator at 4 ℃ for overnight, and carrying out dehydration embedding on the 2 nd day;
h. and (3) cleaning for the second time: washing out redundant fixing solution in the sample by using phosphate buffer solution at the temperature of 4 ℃, wherein the total time is 6 times for 15 minutes for 4 times and 30 minutes for 2 times;
i. third fixation: an osmium tetroxide phosphate buffer solution containing 1% by mass is fixed for 4 hours at 4 ℃;
j. and (3) cleaning for the third time: washing out redundant fixing solution in the sample by using 4 ℃ phosphate buffer solution, wherein the total amount of the fixing solution is 6 times for 15 minutes for 4 times for 30 minutes for 2 times;
k. dehydrating: according to volume fraction, 30% ethanol 20 min-50% ethanol 20 min-70% ethanol 4-80% ethanol 20 min-90% ethanol 20 min-100% ethanol 30 min-propylene oxide 30min 2 times at normal temperature; gradually treating the sample to dehydrate;
l, transition: propylene oxide and EP812 resin are used in a volume ratio of 3:1 for 1 time in 30 minutes at normal temperature; 1:1,1 hour for 1 time, and normal temperature; 1:3,2 hours for 1 time, and gradually treating the sample by the mixed solution at normal temperature;
m, infiltration: soaking the sample with the EPon812 resin for 3 hours and 1 time at normal temperature; soaking the sample with the EPon812 resin for 1 time for 15 hours at normal temperature; soaking the sample with pure EP812 resin for 7 hours for 1 time at normal temperature;
n, embedding: the sample was placed in pure EP812 resin 15 hours 1 time;
o, polymerization: baking the embedded block at 60 ℃ for 60 hours, and solidifying the embedded block to obtain the epoxy resin embedded block, and slicing the epoxy resin embedded block.
The invention firstly combines the primary antibody and the secondary antibody before the conventional sample preparation, so that the problem of activity of the antigen is not needed to be worried, and the problems of poor contact between the antigen and the primary antibody and the secondary antibody are solved by opening a channel on the cell wall and the cell membrane through the educing enzyme and the Triton-100, the success rate of marking is improved, glutaraldehyde and osmium tetroxide can be used in the later steps according to the sample preparation process of the biological tissue block, and the problem that the antigen activity preservation and the tissue structure preservation are difficult to be compatible is solved.
Description of the drawings:
FIG. 1 is a transmission electron microscopic image of a sample obtained by conventional sample preparation of comparative example;
FIG. 2 is a sample obtained by treating with a phosphate buffer solution (containing 0.1% Triton X-100 by mass fraction) in membrane permeation;
FIG. 3 is a sample prepared by membrane permeation treatment with a phosphate buffer solution (containing 0.2% Triton X-100 by mass fraction).
The specific embodiment is as follows:
the following examples are further illustrative of the invention and are not intended to be limiting thereof.
Example 1:
sample information: root nodule of Baimai
Double-fixation embedding sample preparation flow after integral immunization:
1. fixing for the first time: cutting the tissue into small blocks, wherein the maximum thickness is not more than 500 micrometers, and placing the small blocks in a phosphate buffer solution of paraformaldehyde with the mass fraction of 4% for air extraction and preservation for 2 hours at 4 ℃;
2. cleaning for the first time: washing the fixed material with phosphate buffer solution for 5 times, each time for 5min at 4 ℃;
3. digesting the cell wall: placing the tissue block obtained in the step 2 into an enzymolysis solution (containing 0.15% of the educing enzyme Macerozyme,2mM MES, ph 5.0) prepared by a phosphate buffer solution, digesting the material at room temperature for 45min, and then washing 3 times with the phosphate buffer solution at 4 ℃ for 5min each time;
4. membrane permeation: placing the material in the step 3 into a phosphate buffer solution (containing 0.1% Triton X-100 or 0.2% Triton X-100 by mass fraction), treating the material at 4deg.C for 30min, and then washing with phosphate buffer for 5 times each for 5min;
5. blocking (Blocking): soaking the sealing material in 1% (final concentration) skimmed milk powder dissolved in phosphoric acid buffer solution at 4deg.C for 35min;
6. an antibody: incubating the material with primary anti-GFP solution dissolved in phosphate buffer at 4deg.C for 1h;
7. second antibody colloidal gold (Blocking) 4 deg.c: washing the material with phosphate buffer solution at 4deg.C for 5min 3 times each time; incubating the material for 1h with a secondary antibody colloidal gold solution dissolved in a phosphate buffer, and washing the material with the phosphate buffer 5 times for 5min each time;
8. second fixation: re-fixing the material by using a phosphate buffer solution containing 2% of paraformaldehyde and 2.5% of glutaraldehyde by mass, placing the material in a refrigerator at 4 ℃ for overnight, and carrying out dehydration embedding on the 2 nd day;
9. and (3) cleaning for the second time: washing out the excess fixative from the sample with 0.1M phosphate buffer (pH 7.2,4 ℃ C.) for 4 times 15 minutes, 2 times 30 minutes, and 6 times total;
10. third fixation: 0.1M phosphoric acid phosphate buffer solution containing osmium tetroxide with the mass fraction of 1% and the pH of 7.2, and fixing for 4 hours at the temperature of 4 ℃;
11. and (3) cleaning for the third time: washing out the excess fixative in the sample with 0.1M phosphate buffer (pH 7.2, 4deg.C), 15min 4 times 30min 2 times, 6 times altogether, 4deg.C;
12. dehydrating: 30% ethanol 20 minutes to 50% ethanol 20 minutes to 70% ethanol 20 minutes to 80% ethanol 20 minutes to 90% ethanol 20 minutes to 100% ethanol 30 minutes to propylene oxide 30 minutes for 2 times at normal temperature; gradually treating the sample to dehydrate;
13. and (3) transition: gradually treating the sample with a mixed solution of propylene oxide and EP812 resin in a volume ratio of 3:1 (30 min 1 time, normal temperature), 1:1 (1 hour 1 time, normal temperature) and 1:3 (2 hours 1 time, normal temperature);
14. penetration: soaking the sample with the EPon812 resin for 3 hours and 1 time at normal temperature; soaking the sample with the EPon812 resin for 15 hours at normal temperature for 1 time; soaking the sample with pure EP812 resin for 7 hr for 1 time at room temperature
15. Embedding: the sample was placed in pure EP812 resin 15 hours 1 time;
16. polymerization: baking the embedded block at 60 ℃ for 60 hours, solidifying the embedded block to obtain an epoxy resin embedded block, and slicing the epoxy resin embedded block after a week;
17. ultrathin section: slice thickness 70nm;
18. dyeing: 4% uranium acetate staining for 15 minutes.
19. The observation was performed by a transmission electron microscope.
The transmission electron microscope results are shown in fig. 2 and 3.
Comparative example: conventional sample preparation
The comparative example comprises the following steps:
1. fixing: ice bath operation, and preservation at 4 ℃; the mass fraction of 4% paraformaldehyde prepared by 0.1M pH=7.2 sodium phosphate buffer solution and the mass fraction of 0.5% glutaraldehyde fixing solution are used for rapidly cutting small samples, and the sample volume must be smaller than 1mm 3 Pumping and fixing for 2 hours;
2. cleaning: ice bath operation, and preservation at 4 ℃;0.1M ph=7.2 sodium phosphate buffer wash 4 times; each time interval is 15min
3. Dehydrating: ice bath operation; 30% ethanol with volume fraction of 30min, and preserving at 4deg.C; ethanol with volume fraction of 50% for 1h at-20deg.C; ethanol with volume fraction of 70% for 1h at-20deg.C; 100% ethanol for 2 times, each for 1h, at-20deg.C;
4. penetration: ice bath operation, preservation at-35 ℃;100% ethanol: lowicryl K4M (1:1 v/v) for 1 hour; 100% ethanol: K4M 1:2v/v 1 hr; pure K4M twice, one half hour at a time.
5. Embedding: embedding the sample with pure K4M; anaerobic sealing treatment;
6. polymerization: after 48 hours of ultraviolet irradiation at the temperature of minus 35 ℃, the temperature is slowly restored to the normal temperature under the irradiation of weak ultraviolet rays.
7. Ultrathin section: sample cut at 70nm
8. Closing: soaking the closed ultrathin slice for 5min by using 1% (final concentration) of skimmed milk powder dissolved in phosphate buffer solution at 4 ℃;
9. an antibody: incubating the ultrathin sections with primary GFP solution dissolved in phosphate buffer at 4deg.C for 1h;
10. secondary antibody colloidal gold: washing the ultrathin section with phosphate buffer solution at 4 ℃ for 3 times for 5min each time; incubating the ultrathin section for 1h by using a secondary anti-colloidal gold solution dissolved by a phosphate buffer, and soaking the closed ultrathin section twice for 5min each time by using skimmed milk powder with the mass fraction of 1% (final concentration) dissolved by the phosphate buffer; then washing the ultrathin section with phosphate buffer solution for 2 times for 5min each time; ddH 2 O cleaning the ultrathin section for 2 times for 5min each time;
11. dyeing: soaking ultrathin slices in 4% uranyl acetate solution for 5min;
12. cleaning: ddH 2 O cleaning the ultrathin section for 5min for 4 times; air-drying
13. And (5) performing on-machine observation by a transmission electron microscope.
The transmission electron microscope results are shown in FIG. 1.
Description of effects: FIG. 1 shows the results obtained in the comparative examples: the ultrathin section is damaged, the tissue structure of the sample is poor, and no obvious mark (gold particle) is visible; FIG. 2 shows the results of the test example, which shows that triton-100 content is 0.1%: the ultrathin section is smooth and intact, the tissue structure of the sample is good, the fungus membrane is intact, the content is compact and full, the antigen activity is well preserved, the gold particles are well distributed, and the gold particles are fine and smooth and do not get stuck; FIG. 3 shows the results of the test example, which shows that triton-100 content is 0.2%: the ultrathin section is smooth and intact, the tissue structure of the sample is relatively good, the bacterial film is basically intact, the content is slightly lost, the antigen activity is still available, the gold particles are clearly distributed, but the gold particles are slightly clustered. (remark: gold particles are black round compact black dots).
Claims (1)
1. A double-fixation embedding sample preparation method after integral immunization is characterized by comprising the following specific steps:
a. fixing for the first time: cutting the root nodule tissue of the Baimai into small pieces, placing the small pieces in a phosphate buffer solution of paraformaldehyde with the maximum thickness of not more than 500 microns, and carrying out air extraction and preservation for 2 hours at 4 ℃;
b. cleaning for the first time: washing the fixed material with phosphate buffer solution for 5 times, each time for 5min at 4 ℃;
c. digesting the cell wall: preparing an enzymolysis solution containing 0.15% of isolated enzyme by mass percent by using a phosphate buffer solution, placing the tissue block in the step b into the enzymolysis solution, digesting the material for 45min at room temperature, and then washing for 3 times by using the phosphate buffer solution at 4 ℃ for 5min each time;
d. membrane permeation: placing the material in the step c into a phosphoric acid buffer solution containing 0.1% of TritonX-100 by mass, treating the material at 4 ℃ for 30min, and then washing the material with the phosphoric acid buffer solution for 5 times each time for 5min;
e. closing: soaking the sealing material for 35min by using 1% of skimmed milk powder dissolved in phosphoric acid buffer solution at 4 ℃;
f. an antibody: incubating the material for 1h with a primary antibody solution dissolved in phosphate buffer at 4 ℃;
g. secondary antibody colloidal gold: washing the material with phosphate buffer solution at 4deg.C for 5min for 3 times; incubating the material for 1h with a secondary antibody colloidal gold solution dissolved in a phosphate buffer, and washing the material with the phosphate buffer for 5 times, each time for 5min;
h. second fixation: re-fixing the material by using a phosphate buffer solution containing 2% of paraformaldehyde and 2.5% of glutaraldehyde by mass, placing the material in a refrigerator at 4 ℃ for overnight, and carrying out dehydration embedding on the 2 nd day;
i. and (3) cleaning for the second time: washing out redundant fixing solution in the sample by using phosphate buffer solution at the temperature of 4 ℃, wherein the total time is 6 times for 15 minutes for 4 times and 30 minutes for 2 times;
j. third fixation: osmium tetroxide phosphate buffer solution with the mass fraction of 1 percent is fixed for 4 hours at the temperature of 4 ℃;
k. and (3) cleaning for the third time: washing out redundant fixing solution in the sample by using 4 ℃ phosphate buffer solution, wherein the total amount of the fixing solution is 6 times for 15 minutes for 4 times for 30 minutes for 2 times;
and l, dehydration: 30% ethanol for 20 minutes to 50% ethanol for 20 minutes to 70% ethanol overnight in volume fraction, and the above steps are carried out at 4 ℃; the preparation method comprises the following steps of performing at normal temperature, wherein 80% ethanol is used for 20 minutes to 90% ethanol is used for 20 minutes to 100% ethanol is used for 30 minutes to propylene oxide is used for 2 times; gradually treating the sample to dehydrate;
m, transition: propylene oxide and EP812 resin are used in a volume ratio of 3:1 for 1 time in 30 minutes at normal temperature; 1:1,1 hour for 1 time, and normal temperature; 1:3,2 hours for 1 time, and gradually treating the sample by the mixed solution at normal temperature;
n, permeation: soaking the sample with the EPon812 resin for 3 hours and 1 time at normal temperature; soaking the sample with the EPon812 resin for 1 time for 15 hours at normal temperature; soaking the sample with pure EP812 resin for 7 hours for 1 time at normal temperature;
o, embedding: the sample was placed in pure EP812 resin 15 hours 1 time;
p, polymerization: baking the embedded block at 60 ℃ for 60 hours, and solidifying the embedded block to obtain the epoxy resin embedded block, and slicing the epoxy resin embedded block.
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| CN103777001A (en) * | 2014-01-24 | 2014-05-07 | 东南大学 | Hypersensitive immune electron microscope marking method |
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| 杨勇骥 等.胶体金标记免疫电镜技术.《实用生物医学电子显微镜技术》.第二军医大学出版社,2003,第119-121页. * |
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