CN113418766A - Double-fixation embedding sample preparation method after whole immunization - Google Patents
Double-fixation embedding sample preparation method after whole immunization Download PDFInfo
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- CN113418766A CN113418766A CN202110703166.XA CN202110703166A CN113418766A CN 113418766 A CN113418766 A CN 113418766A CN 202110703166 A CN202110703166 A CN 202110703166A CN 113418766 A CN113418766 A CN 113418766A
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- 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
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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
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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
- G01N23/02—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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- 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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Abstract
The invention discloses a double-fixation embedding sample preparation method after integral immunization. In the method, a primary antibody and a secondary antibody are combined with an antigen before the conventional sample preparation of a transmission electron microscope sample, and then the sample preparation of the transmission electron microscope sample is carried out. In the invention, the primary-antibody and the secondary-antibody are combined with the antigen in front of the conventional sample preparation, so that the activity problem of the antigen does not need to be worried, a channel is opened on a cell wall and a cell membrane through the eductase and the Triton-100, the problem of poor contact between the antigen and the primary-antibody and the secondary-antibody is solved, the success rate of marking is improved, and glutaraldehyde and osmium tetroxide can be used in the later step according to the sample preparation process of a biological tissue block, so that the problem that the antigen activity preservation and the tissue structure preservation are difficult to be compatible is solved.
Description
The technical field is as follows:
the invention belongs to the field of transmission electron microscopes, and particularly relates to a double-fixation embedding sample preparation method after integral immunization.
Background art:
the transmission electron microscope is the microscope with the highest resolution. The transmission electron microscope can be used for observing the ultrastructure of the tissue, and the expected distribution condition of enzyme, specific ion and antigen (protein in most cases) in the organelle can be known by combining the enzyme positioning, ion positioning and immune nano gold labeling technologies.
Transmission electron microscopy requires that the sample be very thin and free of water, so biological tissues usually need to undergo the process of aldehyde fixation-osmium tetroxide fixation-dehydration-embedding-ultrathin sectioning-heavy metal staining before being observed in the transmission electron microscope. In the conventional immunolabeling technology, a primary antibody is used for binding antigen after the ultrathin section is prepared, and a secondary antibody containing nano gold particles is used for binding the primary antibody. The gold particles are imaged as black dots, which is different from the tissue imaging result, so that the distribution of the antigen in the cells can be obtained (the antigen is marked to obtain the positioning information thereof). Because the glutaraldehyde and the osmium tetroxide in the aldehyde group fixing solution can completely denature the antigen and cannot be combined with the primary antibody, two fixing agents, namely the glutaraldehyde and the osmium tetroxide, are not used in the subsequent preparation process of the sample needing to be labeled by the immunogold, and the antigen activity is ensured by sacrificing the fixing effect of the tissue structure. However, fixation is a very critical step in sample preparation, and aims to terminate the biochemical process of cells, stabilize the components of cell substances, maintain the original fine structure of living cells, avoid cell autolysis and putrefaction caused by invasion of external microorganisms, cause the appearance of false images, and establish cross-linking between molecules of some components through chemical reaction or physical reaction to provide a skeleton to stabilize the spatial configuration of various organelles, and the fixing agent osmium tetroxide can also improve the image contrast effect. Because only paraformaldehyde is used as a fixing agent, an immune labeled sample is usually not well fixed, the finally presented tissue structure is poor, even an antigen is very sensitive and easy to deform, and the tissue structure and the antigen activity in the whole experimental process cannot be well preserved.
Disclosure of Invention
The invention aims to provide a double-fixation embedding sample preparation method after integral immunization, which has intact antigen activity preservation and tissue structure preservation.
The invention relates to a sample preparation method of integral immunization and double fixation embedding, which is characterized in that a primary antibody and a secondary antibody are combined with antigen before the conventional sample preparation (aldehyde fixation-osmium tetroxide fixation-dehydration-embedding) of a transmission electron microscope sample, and then the sample preparation of the transmission electron microscope sample is carried out.
Preferably, the immobilized material is treated with an eductase and/or Triton-100 prior to the primary antibody binding to the antigen, such that the cell walls and membranes of the material are opened.
Preferably, the specific steps of treating the immobilized material with an eductase and/or Triton-100 before the primary antibody binds to the antigen are: cutting the tissue into blocks, fixing, putting the material washed by the phosphate buffer solution into the resolution enzyme enzymolysis solution for digestion, then washing, putting the material into the Triton X-100 solution for treatment, and then washing the material by the phosphate buffer solution.
More preferably, the macerozyme is 0.15% by mass, and the TritonX-100 solution is 0.1-0.2% by mass.
Preferably, the method comprises the following specific steps:
A. cutting the tissue into blocks, fixing the blocks by using paraformaldehyde with the mass fraction of 4%, digesting cell walls by using 0.15% of segregation enzymatic hydrolysate, soaking a sealing material by using 1% of skimmed milk powder, and then sequentially carrying out primary antibody incubation and secondary antibody incubation;
B. and (3) fixing the incubated material by using a solution containing 2 mass percent of paraformaldehyde and 2.5 mass percent of glutaraldehyde, fixing the incubated material by using 1 mass percent of osmium tetroxide, cleaning the fixed material, and dehydrating, transiting, permeating, embedding and polymerizing to obtain the epoxy resin embedded block.
The epoxy resin embedded block can be observed by a transmission electron microscope after ultrathin slicing and dyeing.
Further preferably, the method comprises the following specific steps:
a. first fixing: cutting the tissue into small pieces, wherein the maximum thickness is not more than 500 micrometers, and placing the small pieces in a phosphate buffer solution of paraformaldehyde with the mass fraction of 4% for air suction and storage 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, 4 deg.C;
c. cell wall digestion: b, preparing an enzymolysis solution containing 0.15 mass percent of the eductase 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 the material for 5min each time for 3 times by using the phosphate buffer solution at 4 ℃;
d. and (3) membrane permeation: c, placing the material in the step c into a phosphoric acid buffer solution containing 0.1-0.2% of TritonX-100 by mass percent, treating the material for 30min at 4 ℃, and then washing the material for 5 times and 5min each time by using the phosphoric acid buffer solution;
e. and (3) sealing: soaking the sealing material in 1 wt% skimmed milk powder dissolved in phosphate buffer at 4 deg.C for 35 min;
f. a first antibody: incubating the material with an anti-solution dissolved in a phosphate buffer at 4 ℃ for 1 h;
g. secondary antibody colloidal gold: washing the material with phosphate buffer solution at 4 deg.C for 5min for 3 times; incubating the material with a secondary antibody colloidal gold solution dissolved in a phosphate buffer solution for 1h, and washing the material with the phosphate buffer solution for 5 times, each time for 5 min;
e. and (3) second fixing: fixing the material again by using a phosphate buffer solution containing 2 mass percent of paraformaldehyde and 2.5 mass percent of glutaraldehyde, putting the material in a refrigerator at 4 ℃ overnight, and dehydrating and embedding the material after 2 days;
h. and (3) cleaning for the second time: washing off excessive fixative in the sample with 4 deg.C phosphate buffer solution for 6 times (4 times in 15min and 2 times in 30 min);
i. and (3) third fixation: containing 1% by mass of osmium tetroxide phosphate buffer solution, and fixing for 4 hours at 4 ℃;
j. and (3) cleaning for the third time: washing off excessive fixative in the sample with 4 deg.C phosphate buffer solution for 6 times, and for 15min, 4 times, 30min and 2 times;
k. and (3) dehydrating: according to volume fraction, 30% ethanol is used for 20 minutes to 50% ethanol for 20 minutes to 70% ethanol, 4% ethanol to 80% ethanol is used for 20 minutes to 90% ethanol for 20 minutes to 100% ethanol for 30 minutes to propylene oxide for 30 minutes and 2 times at normal temperature; gradually treating the sample for dehydration;
l, transition: the volume ratio of the propylene oxide to the EP812 resin is 3:1, the time is 1 in 30 minutes, and the temperature is normal; 1:1, 1 hour and 1 time, and normal temperature; 1:3, 2 hours and 1 time, and gradually treating the sample by the normal-temperature mixed solution;
m, penetration: soaking the sample in EPon812 resin for 3 hours and 1 time at normal temperature; then soaking the sample in EPon812 resin for 15 hours and 1 time, and keeping the temperature at normal temperature; soaking the sample in pure EP812 resin for 7 hours and 1 time at normal temperature;
n, embedding: the samples were placed in neat EP812 resin for 15 hours 1 time;
o, polymerization: and (3) baking the embedding block for 60 hours at 60 ℃ to solidify the embedding block to obtain the epoxy resin embedding block to be sliced.
In the invention, the primary-antibody and the secondary-antibody are combined with the antigen in front of the conventional sample preparation, so that the activity problem of the antigen does not need to be worried, a channel is opened on a cell wall and a cell membrane through the eductase and the Triton-100, the problem of poor contact between the antigen and the primary-antibody and the secondary-antibody is solved, the success rate of marking is improved, and glutaraldehyde and osmium tetroxide can be used in the later step according to the sample preparation process of a biological tissue block, so that 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 micrograph of a sample obtained by a conventional sample preparation of a comparative example;
FIG. 2 is a diagram showing a prepared sample treated with a phosphate buffer solution (containing 0.1% Triton X-100 by mass) in membrane permeation;
FIG. 3 shows a prepared sample treated with a phosphate buffer solution (containing 0.2% Triton X-100 by mass) in membrane permeation.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1:
sample information: root nodule of Lotus vein
A double-fixation embedding sample preparation process after integral immunization:
1. first fixing: cutting the tissue into small pieces, wherein the maximum thickness is not more than 500 micrometers, and placing the small pieces in a phosphate buffer solution of paraformaldehyde with the mass fraction of 4% for air suction and storage 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, 4 deg.C;
3. cell wall digestion: placing the tissue block obtained in the step 2 in an enzymolysis solution (containing a final concentration of 0.15% of an eductase macrozyme, 2mM MES and ph5.0 by mass fraction), digesting the material at room temperature for 45min, and then washing the material for 5min each time for 3 times by using a phosphoric acid buffer solution at 4 ℃;
4. and (3) membrane permeation: placing the material obtained in the step (3) in a phosphate buffer solution (containing 0.1% TritonX-100 or 0.2% TritonX-100 by mass fraction), treating the material at 4 ℃ for 30min, and then washing the material 5 times for 5min each time by using the phosphate buffer;
5. blocking (Blocking): soaking the sealing material in skimmed milk powder with a mass fraction of 1% (final concentration) dissolved in phosphate buffer at 4 deg.C for 35 min;
6. a first antibody: incubating the material with a primary anti-GFP solution dissolved in phosphate buffer at 4 ℃ for 1 h;
7. secondary antibody colloidal gold (Blocking)4 ℃: washing the material with phosphate buffer solution at 4 deg.C for 5min 3 times; incubating the material with a secondary antibody colloidal gold solution dissolved in a phosphate buffer solution for 1h, and washing the material with the phosphate buffer solution for 5 times and 5min each time;
8. and (3) second fixing: fixing the material again by using a phosphate buffer solution containing 2 mass percent of paraformaldehyde and 2.5 mass percent of glutaraldehyde, putting the material in a refrigerator at 4 ℃ overnight, and dehydrating and embedding the material after 2 days;
9. and (3) cleaning for the second time: washing off excessive fixative in the sample with 0.1M phosphate buffer (pH7.2, 4 deg.C) for 6 times (4 times in 15min, 2 times in 30 min);
10. and (3) third fixation: osmium tetroxide with the mass fraction of 1 percent and 0.1M phosphate buffer solution with the PH value of 7.2 are fixed for 4 hours at the temperature of 4 ℃;
11. and (3) cleaning for the third time: washing off excess fixative in the sample with 0.1M phosphate buffer (pH7.2, 4 deg.C), 15min 4 times 30min 2 times, 6 times, 4 deg.C;
12. and (3) dehydrating: according to volume fraction, 30% ethanol is used for 20 minutes to 50% ethanol for 20 minutes to 70% ethanol, the mixture is taken overnight for 4 to 80% ethanol for 20 minutes to 90% ethanol for 20 minutes to 100% ethanol for 30 minutes to propylene oxide for 30 minutes and then is subjected to normal temperature for 2 times; gradually treating the sample for dehydration;
13. and (3) transition: gradually treating the sample with a mixed solution of 3:1(30 minutes and 1 time at normal temperature), 1:1(1 hour and 1 time at normal temperature) and 1:3(2 hours and 1 time at normal temperature) by volume ratio of the propylene oxide to the EP812 resin;
14. and (3) infiltration: soaking the sample in EPon812 resin for 3 hours and 1 time at normal temperature; then soaking the sample in EPon812 resin for 15 hours and 1 time at normal temperature; soaking the sample with pure EP812 resin for 7 hours and 1 time at normal temperature
15. Embedding: the samples were placed in neat EP812 resin for 15 hours 1 time;
16. polymerization: baking the embedding block for 60 hours at 60 ℃ to solidify the embedding block to obtain an epoxy resin embedding block, and standing the epoxy resin embedding block for a week to be sliced;
17. ultrathin slicing: the slice thickness is 70 nm;
18. dyeing: staining with 4% uranium acetate for 15 minutes.
19. Observation was performed by transmission electron microscopy.
The transmission electron microscope results are shown in fig. 2 and 3.
Comparative example: preparing a sample by a conventional method
The comparative example comprises the following steps:
1. fixing: performing ice-bath operation, and storing at 4 ℃; 0.1M pH7.2 sodium phosphate buffer solution preparation with mass fraction of 4% polymethyl methacrylateQuickly cutting a sample in aldehyde and glutaraldehyde fixing solution with the mass fraction of 0.5%, wherein the volume of the sample is required to be less than 1mm3Pumping air and fixing for 2 hours;
2. cleaning: performing ice-bath operation, and storing at 4 ℃; washing 4 times with 0.1M pH7.2 sodium phosphate buffer; each time at intervals of 15min
3. And (3) dehydrating: performing ice bath operation; ethanol with volume fraction of 30% for 30min, and storing at 4 deg.C; storing with 50% ethanol by volume for 1h at-20 deg.C; storing with 70% ethanol for 1h at-20 deg.C; storing with 100% ethanol for 2 times, each for 1 hr at-20 deg.C;
4. and (3) infiltration: ice-bath operation and preservation at-35 ℃; 100% ethanol: lowicryl K4M (1:1v/v) for 1 hour; 100% ethanol: K4M 1:2v/v 1 hour; pure K4M was used twice, one and a half hours each time.
5. Embedding: embedding the sample with pure K4M; anaerobic sealing treatment;
6. polymerization: after ultraviolet irradiation at-35 ℃ for 48h, the temperature is slowly recovered to the normal temperature under the weak ultraviolet irradiation.
7. Ultrathin slicing: cutting the sample to 70nm
8. And (3) sealing: soaking the sealed ultrathin slice with 1% (final concentration) of skimmed milk powder dissolved in phosphate buffer at 4 deg.C for 5 min;
9. a first antibody: incubating the ultrathin section with a primary anti-GFP solution dissolved in a phosphate buffer at 4 ℃ for 1 h;
10. secondary antibody colloidal gold: washing the ultrathin slice with phosphate buffer solution at 4 deg.C for 5min 3 times each time; incubating the ultrathin section for 1h by using a secondary antibody colloidal gold solution dissolved in a phosphate buffer solution, and soaking the sealed ultrathin section twice for 5min by using skim milk powder with the mass fraction of 1% (final concentration) dissolved in the phosphate buffer solution; then washing the ultrathin slice with phosphate buffer solution for 5min 2 times each time; ddH2Cleaning the ultrathin slice for 5min 2 times each time;
11. dyeing: soaking the ultrathin slice in 4% uranyl acetate solution for 5 min;
12. cleaning: ddH2Cleaning the ultrathin slice for 5min 4 times each time; air drying
13. And (5) observing on a computer by using a transmission electron microscope.
The transmission electron microscopy results are shown in FIG. 1.
Description of the effects: FIG. 1 shows the results obtained in the comparative example: the ultrathin section is damaged, the tissue structure of the sample is poor, and no obvious mark (gold particles) is seen; FIG. 2 shows the results of the experimental example in which the triton-100 content was 0.1%: the ultrathin section is flat and intact, the tissue structure of the sample is good, the mycoderm is intact, the inclusion is compact and full, the activity of the antigen is well preserved, the gold particles are clearly distributed, and the ultrathin section is fine and smooth and does not agglomerate; FIG. 3 shows the results of the experimental example with a triton-100 content of 0.2%: the ultrathin section is flat and intact, the tissue structure of the sample is good, the mycoderm is basically intact, the inclusion is slightly lost, the antigen activity is still acceptable, the gold particles are clearly distributed, but are slightly agglomerated. (Note: the gold particles are black round dense black dots).
Claims (6)
1. A method for preparing specimen by embedding and double-fixing after integral immunization is characterized in that a primary antibody and a secondary antibody are combined with antigen before the conventional specimen preparation of a transmission electron microscope specimen, and then the specimen preparation of the transmission electron microscope specimen is carried out.
2. The method of claim 1, wherein the immobilized material is treated with an eductase and/or Triton-100 before the primary antibody binds to the antigen, so that the cell wall and cell membrane of the material are opened.
3. The method of claim 2, wherein the step of treating the immobilized material with an eductase and/or Triton-100 before the primary antibody binds to the antigen comprises: cutting the tissue into blocks, fixing, putting the material washed by the phosphate buffer solution into the resolution enzyme enzymolysis solution for digestion, then washing, putting the material into the Triton X-100 solution for treatment, and then washing the material by the phosphate buffer solution.
4. The double immobilization embedding sample preparation method after whole body immunization according to claim 3, wherein the isolation enzyme is 0.15% by mass fraction isolation enzyme, and the TritonX-100 solution is 0.1% to 0.2% by mass fraction TritonX-100 solution.
5. The method for preparing the double-fixed embedded sample after the integral immunization according to claim 1 is characterized by comprising the following specific steps:
a. cutting the tissue into blocks, fixing the blocks by using paraformaldehyde with the mass fraction of 4%, digesting cell walls by using 0.15% of segregation enzymatic hydrolysate, soaking a sealing material by using 1% of skimmed milk powder, and then sequentially carrying out primary antibody incubation and secondary antibody incubation;
b. and (3) fixing the incubated material by using a solution containing 2 mass percent of paraformaldehyde and 2.5 mass percent of glutaraldehyde, fixing the incubated material by using 1 mass percent of osmium tetroxide, cleaning the fixed material, and dehydrating, transiting, permeating, embedding and polymerizing to obtain the epoxy resin embedded block.
6. The method for preparing the double-fixed embedded sample after the whole immunization according to claim 1 or 5, which is characterized by comprising the following specific steps:
a. first fixing: cutting the tissue into small pieces, wherein the maximum thickness is not more than 500 micrometers, and placing the small pieces in a phosphate buffer solution of paraformaldehyde with the mass fraction of 4% for air suction and storage 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, 4 deg.C;
c. cell wall digestion: b, preparing an enzymolysis solution containing 0.15 mass percent of the eductase 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 the material for 5min each time for 3 times by using the phosphate buffer solution at 4 ℃;
d. and (3) membrane permeation: c, 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 for 30min at 4 ℃, and then washing the material for 5 times and 5min each time by using the phosphate buffer solution;
e. and (3) sealing: soaking the sealing material in 1 wt% skimmed milk powder dissolved in phosphate buffer at 4 deg.C for 35 min;
f. a first antibody: incubating the material with an anti-solution dissolved in a phosphate buffer at 4 ℃ for 1 h;
g. secondary antibody colloidal gold: washing the material with phosphate buffer solution at 4 deg.C for 5min for 3 times; incubating the material with a secondary antibody colloidal gold solution dissolved in a phosphate buffer solution for 1h, and washing the material with the phosphate buffer solution for 5 times, each time for 5 min;
e. and (3) second fixing: fixing the material again by using a phosphate buffer solution containing 2 mass percent of paraformaldehyde and 2.5 mass percent of glutaraldehyde, putting the material in a refrigerator at 4 ℃ overnight, and dehydrating and embedding the material after 2 days;
h. and (3) cleaning for the second time: washing off excessive fixative in the sample with 4 deg.C phosphate buffer solution for 6 times (4 times in 15min and 2 times in 30 min);
i. and (3) third fixation: containing 1% by mass of osmium tetroxide phosphate buffer solution, and fixing for 4 hours at 4 ℃;
j. and (3) cleaning for the third time: washing off excessive fixative in the sample with 4 deg.C phosphate buffer solution for 6 times, and for 15min, 4 times, 30min and 2 times;
k. and (3) dehydrating: according to volume fraction, 30% ethanol is used for 20 minutes to 50% ethanol for 20 minutes to 70% ethanol, 4% ethanol to 80% ethanol is used for 20 minutes to 90% ethanol for 20 minutes to 100% ethanol for 30 minutes to propylene oxide for 30 minutes and 2 times at normal temperature; gradually treating the sample for dehydration;
l, transition: the volume ratio of the propylene oxide to the EP812 resin is 3:1, the time is 1 in 30 minutes, and the temperature is normal; 1:1, 1 hour and 1 time, and normal temperature; 1:3, 2 hours and 1 time, and gradually treating the sample by the normal-temperature mixed solution;
m, penetration: soaking the sample in EPon812 resin for 3 hours and 1 time at normal temperature; then soaking the sample in EPon812 resin for 15 hours and 1 time, and keeping the temperature at normal temperature; soaking the sample in pure EP812 resin for 7 hours and 1 time at normal temperature;
n, embedding: the samples were placed in neat EP812 resin for 15 hours 1 time;
o, polymerization: and (3) baking the embedding block for 60 hours at 60 ℃ to solidify the embedding block to obtain the epoxy resin embedding block to be sliced.
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| CN1811433A (en) * | 2006-02-20 | 2006-08-02 | 中国科学院植物研究所 | Method for detecting gymnosperm pollen in-tube antigen |
| CN103777001A (en) * | 2014-01-24 | 2014-05-07 | 东南大学 | Hypersensitive immune electron microscope marking method |
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