Immunogold colloidal gold electron microscopy detection method based on spurr embedding plant tissue sample ultrathin section

Abstract

The application provides an immunocolloidal gold electron microscope detection method based on Spurr embedding plant tissue samples and ultrathin sections, and belongs to the technical field of plant tissue embedding. The application provides application of Spurr as an embedding agent of plant tissues to ultrathin sections in immunocolloidal gold electron microscope detection. In the immunocolloidal gold electron microscope detection provided by the application, the plant tissues are embedded by using the Spurr embedding agent, plant tissue ultrathin sections are obtained, and then the plant tissue ultrathin sections are treated by using an immunocolloidal gold labeling method and then detected, so that a detection result with clear virus morphology and cell ultrastructure and high gold particle contrast can be obtained, and false positives are not prone to occurring in the detection result.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue embedding technology, specifically relating to an immunochromatographic method for detecting ultrathin sections of plant tissue samples embedded in Spurr using colloidal gold electron microscopy. Background Technology

[0002] Immunocolloidal gold technology is an immunolabeling technique that uses colloidal gold to generate color and specifically react with antigens to achieve detection. With the increasing maturity of antibody technology, immunocolloidal gold technology has also been gradually improved and developed. By combining with techniques such as electron microscopy, immunochromatography, and percolation, immunocolloidal gold technology has successfully detected bioactive substances such as proteins, hormones, and cytokines. Immunocolloidal gold electron microscopy is a combination of immunocolloidal gold technology and electron microscopy. It observes and studies the morphology and function of tissue cells at the ultrastructural level by binding labeled antibodies with specific antigens. In immunocolloidal gold electron microscopy, the primary antibody is an antibody that specifically binds to the target protein, and the secondary antibody is an antibody labeled with appropriately sized gold particles that matches the origin of the primary antibody. Gold particles have a high electron density under an electron microscope and are clearly visible. Therefore, the colloidal gold-labeled secondary antibody binds to the corresponding primary antibody, thereby labeling the target protein. In recent years, immunocolloidal gold electron microscopy has been widely used in medical pathology diagnosis (nephropathy, tumors, neuropathy, virus detection, etc.). Although its application in the field of botany is not as extensive as in the field of medical biology, in recent years, immunochromatographic gold electron microscopy has been fully applied to the diagnosis and detection of plant viruses, and it is also gradually being utilized in the interaction between plants and exogenous organisms.

[0003] Currently, the embedding agents successfully used in immunogold electron microscopy include Epon812, LRwhite, and K4M. However, these embedding agents often result in false positives and poor image clarity when applied to immunogold electron microscopy. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide the application of Spurr as an embedding agent for plant tissues in ultrathin sections for immunogold electron microscopy. Using Spurr as an embedding agent for plant tissues in immunogold electron microscopy can yield clear immunogold electron microscopy results with high gold particle contrast.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] This invention provides the application of Spurr as an embedding agent for plant tissues in ultrathin sections for immunocolloidal gold electron microscopy.

[0007] This invention provides an immunogold electron microscopy method for detecting ultrathin sections of Spurr-embedded plant tissue samples, comprising the following steps:

[0008] Plant tissues were embedded using Spurr embedding medium to obtain ultrathin sections of plant tissue;

[0009] Immunogold electron microscopy was used to examine ultrathin sections of plant tissue.

[0010] Preferably, after obtaining ultrathin sections of plant tissue, the method for immunogold electron microscopy detection includes the following steps:

[0011] Plant tissue ultrathin sections are eroded and sealed to obtain sealed ultrathin sections;

[0012] The blocked ultrathin sections were incubated with primary antibody to obtain primary antibody ultrathin sections;

[0013] The primary antibody ultrathin sections were incubated with gold-labeled secondary antibody to obtain gold-labeled secondary antibody ultrathin sections;

[0014] The ultrathin sections of gold-labeled secondary antibody were stained to obtain immunogold-labeled ultrathin sections of plant tissue, which were then examined under an electron microscope.

[0015] Preferably, the incubation temperature of the primary antibody is 20℃~28℃; the incubation time of the primary antibody is 1~2h; the incubation temperature of the secondary antibody is 20℃~28℃; and the incubation time of the secondary antibody is 1~2h.

[0016] Preferably, the sealing includes: sealing ultrathin sections of plant tissue with a 1% (w / w) BSA aqueous solution;

[0017] The temperature of the sealing treatment is 20℃~28℃; the sealing treatment time is 1~2h.

[0018] Preferably, the method for preparing the ultrathin sections of plant tissue includes the following steps:

[0019] Plant tissues are fixed to obtain fixed tissues;

[0020] After dehydration, the fixed tissue was embedded using Spurr embedding medium to obtain embedded tissue.

[0021] The embedded tissue is polymerized to obtain polymerized embedded blocks; the polymerization temperature is 60℃~70℃ and the polymerization time is 12~24h.

[0022] The polymer-embedded blocks were subjected to ultrathin sectioning to obtain ultrathin sections of plant tissue.

[0023] Preferably, the fixation includes a first fixation and a second fixation; the first fixation is performed using a glutaraldehyde aqueous solution with a volume percentage of 2.5% and the fixation time is 24-48 hours; the second fixation is performed using an osmium tetroxide aqueous solution with a volume percentage of 1% and the fixation time is 1.5-2 hours.

[0024] Preferably, the dehydration is performed by gradually dehydrating with ethanol aqueous solutions of different concentrations, and finally by dehydrating with anhydrous ethanol to obtain dehydrated plant tissue; the ethanol aqueous solutions of different concentrations include 30% (v / v) ethanol aqueous solution, 50% (v / v) ethanol aqueous solution, 70% (v / v) ethanol aqueous solution, 80% (v / v) ethanol aqueous solution, 90% (v / v) ethanol aqueous solution and 95% (v / v) ethanol aqueous solution; the anhydrous ethanol dehydration treatment is performed twice.

[0025] Preferably, a gradient infiltration method is used for gradual encapsulation; the gradual encapsulation process includes:

[0026] Dehydrated plant tissue was soaked in acetone to obtain acetone-soaked plant tissue; the soaking time was 20-30 minutes.

[0027] Plant tissues soaked in acetone were first embedded in a mixture of Spurr embedding agent and acetone at a volume ratio of 1:1 to obtain the first embedded product; the first embedding time was 1 to 1.5 hours.

[0028] The first embedded product was subjected to a second embedding in a mixture of Spurr embedding agent and acetone at a volume ratio of 3:1 to obtain the second embedded product; the second embedding time was 3 to 3.5 hours.

[0029] The second embedding product was then embedded a third time in Spurr embedding medium to obtain plant tissue embedding blocks; the third embedding time was 12-15 hours.

[0030] Preferably, the staining method includes uranium acetate staining and lead citrate staining.

[0031] The beneficial effects of this invention are:

[0032] This invention provides the application of Spurr as an embedding agent for plant tissue in ultrathin sections for immunogold electron microscopy. In the immunogold electron microscopy provided by this invention, plant tissue is embedded using Spurr to obtain ultrathin sections. These ultrathin sections are then processed using an immunogold labeling method before detection. This method yields detection results with clear viral morphology and cellular ultrastructure, high gold particle contrast, and a low likelihood of false positives. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is a diagram showing the detection results of immunogold electron microscopy in Example 1;

[0035] Figure 2 This is a comparison image showing the results of immunogold electron microscopy for Example 1.

[0036] Figure 3 This is a diagram showing the detection results of immunogold electron microscopy in Example 2;

[0037] Figure 4 This is a comparison image showing the results of immunogold electron microscopy for Example 2.

[0038] Figure 5 This is a comparison example 3, showing the results of immunogold electron microscopy.

[0039] Figure 6 This is a comparison image showing the results of immunogold electron microscopy for Example 4.

[0040] Figure 7 This is a comparison image showing the results of immunogold electron microscopy for Example 5.

[0041] Figure 8 The image shows the results of immunogold electron microscopy for Comparative Example 6. Detailed Implementation

[0042] This invention provides the application of Spurr as an embedding agent for plant tissues in ultrathin sections for immunocolloidal gold electron microscopy.

[0043] This invention uses Spurr embedding agent to embed plant tissue to obtain ultrathin sections of plant tissue. Then, the ultrathin sections of plant tissue are examined by immunochromatographic gold electron microscopy, which can obtain detection results with clear virus morphology and cell ultrastructure, high contrast of gold particles, and the detection results are less likely to produce false positives.

[0044] This invention provides an immunogold electron microscopy method for detecting ultrathin sections of Spurr-embedded plant tissue samples, comprising the following steps:

[0045] Plant tissues were embedded using Spurr embedding medium to obtain ultrathin sections of plant tissue;

[0046] Immunogold electron microscopy was used to examine ultrathin sections of plant tissue.

[0047] This invention uses Spurr embedding agent to embed plant tissues to obtain ultrathin sections of plant tissues.

[0048] In this invention, the embedding is performed using Spurr embedding medium. This invention does not have any particular limitation on the type of plant tissue; any conventional plant tissue in the art can be used. In this invention, the plant tissue is preferably fresh plant tissue, more preferably fresh leaf tissue. This invention preferably uses the model plant tobacco as an example, embedding tobacco leaves to illustrate the immunogold electron microscopy detection method for ultrathin sections of Spurr-embedded plant tissue samples.

[0049] In this invention, the method for preparing the ultrathin sections of plant tissue preferably includes the following steps:

[0050] Plant tissues are fixed to obtain fixed tissues;

[0051] After dehydration, the fixed tissue was embedded using Spurr embedding medium to obtain embedded tissue.

[0052] The embedded tissue was polymerized to obtain polymerized embedded blocks;

[0053] The polymer-embedded blocks were subjected to ultrathin sectioning to obtain ultrathin sections of plant tissue.

[0054] The present invention preferably involves fixing plant tissue to obtain fixed tissue.

[0055] Before fixing the plant tissue, it is preferable to cut the plant tissue into pieces. Preferably, the plant tissue is cut into 1×2mm pieces. 2 Small pieces are obtained to form tissue blocks. The present invention does not specifically limit the method of slicing; any conventional slicing method in the art can be used. Preferably, a Gillette blade is used for slicing, and more preferably, a newly opened Gillette blade is used. After obtaining the tissue blocks, the present invention preferably fixes the tissue blocks.

[0056] In this invention, the fixation preferably includes a first fixation and a second fixation. The fixation is preferably performed in a centrifuge tube; the centrifuge tube preferably includes a 2.0 mL centrifuge tube.

[0057] In this invention, the first fixation is preferably performed using a glutaraldehyde aqueous solution with a volume percentage of 2.5%. Preferably, the glutaraldehyde aqueous solution is a glutaraldehyde-PBS aqueous solution; the molar concentration of PBS in the glutaraldehyde-PBS aqueous solution is preferably 0.05M. This invention uses a 2.5% volume percentage glutaraldehyde aqueous solution for the first fixation of plant tissue to prevent insufficient fixation due to excessively low concentration, while also preventing excessively high concentration from affecting the internal structure of leaf cells. This invention does not have a special limitation on the amount of glutaraldehyde solution used, as long as the glutaraldehyde solution exceeds the cut tissue and completely covers it. In this invention, when four cut tissues are used for the first fixation, the amount of glutaraldehyde solution used is preferably 600–1000 μL, more preferably 1000 μL. After mixing the cut tissue and the glutaraldehyde solution, this invention preferably performs vacuum filtration on the first fixation reaction system. This invention does not have a special limitation on the vacuum filtration method; any conventional vacuum filtration method in the art can be used. The vacuum filtration time described in this invention is preferably 10-20 minutes, more preferably 10 minutes. The vacuum filtration of the first fixation reaction system in this invention primarily ensures that the tissue fragments sink to the bottom of the glutaraldehyde solution, preventing the leaves from floating on the surface and causing insufficient tissue fixation. After vacuum filtration, the invention performs a first fixation. The first fixation time is preferably 24-48 hours, more preferably 24 hours; the first fixation temperature is preferably 4℃-8℃, more preferably 4℃. After the first fixation, a first fixed leaf is obtained. After obtaining the first fixed leaf, the invention preferably performs a first rinsing. The rinsing solution is preferably 0.1M PBS. The number of rinsings is preferably 3 times; the rinsing time for each rinsing is preferably 15 minutes.

[0058] After the first rinsing is completed, the present invention preferably performs a second fixation on the blades after the first rinsing.

[0059] In this invention, the second fixation is preferably performed using an osmium tetroxide aqueous solution with a volume percentage of 1%. There are no special limitations on the amount of the osmium tetroxide aqueous solution used, as long as it completely covers the cut tissue. In this invention, when four tissue pieces are subjected to the second fixation, the amount of the osmium tetroxide aqueous solution is preferably 200–250 μL, more preferably 200 μL. In this invention, the rinsed leaf and the osmium tetroxide aqueous solution are preferably mixed before the second fixation. In this invention, the second fixation time is preferably 1.5–2 hours, more preferably 2 hours; the second fixation temperature is preferably 20°C–28°C, more preferably 24°C. After the second fixation is completed, a second-fixed leaf is obtained.

[0060] This invention uses a 1% osmium tetroxide (OST) aqueous solution for fixation to maintain the ultrastructure and electron microscopic contrast of cells. OST can better fix the structure of organelles, mainly lipids and fats, and also has a staining effect (appropriately improving contrast) to facilitate electron microscopic observation (without OST fixation, electron micrographs may have poor black-and-white contrast, unclear organelle structures, and poor contrast). Too high a concentration will result in excessively high electron density, while too low a concentration will lead to poor fixation and penetration. Therefore, this invention uses a 1% (v / v) OST aqueous solution for fixation. Furthermore, prolonged OST fixation will make the tissue very hard, making subsequent sectioning and other operations difficult.

[0061] After obtaining the second fixed leaflet, the present invention preferably performs a second rinsing on the second fixed leaflet. The rinsing solution used in the present invention is preferably 0.1M PBS. The number of rinsing cycles is preferably three; the rinsing time for each cycle is preferably 15 minutes. After the second rinsing is completed, the present invention preferably obtains fixed tissue.

[0062] After obtaining the fixed tissue, the present invention preferably dehydrates the fixed tissue and embeds it with Spurr embedding agent to obtain embedded tissue.

[0063] After obtaining the fixed tissue, the present invention preferably dehydrates the fixed tissue. The dehydration in the present invention preferably involves gradual dehydration with ethanol aqueous solutions of different concentrations, followed by final dehydration with anhydrous ethanol to obtain dehydrated plant tissue. In the present invention, the ethanol aqueous solutions of different concentrations preferably include 30% (v / v) ethanol aqueous solution, 50% (v / v) ethanol aqueous solution, 70% (v / v) ethanol aqueous solution, 80% (v / v) ethanol aqueous solution, 90% (v / v) ethanol aqueous solution, and 95% (v / v) ethanol aqueous solution. In the present invention, the fixed tissue is preferably subjected to a first dehydration in a 30% (v / v) ethanol aqueous solution to obtain a first dehydrated product. In the present invention, the first dehydration time is preferably 10-15 minutes, more preferably 15 minutes. After the first dehydration is completed, the present invention preferably involves a second dehydration in a 50% (v / v) ethanol aqueous solution to obtain a second dehydrated product. In the present invention, the second dehydration time is preferably 10-15 minutes, more preferably 15 minutes. After the second dehydration, the present invention preferably performs a third dehydration on the second dehydrated product in a 70% (v / v) ethanol aqueous solution to obtain a third dehydrated product. In the present invention, the third dehydration time is preferably 10-15 min, more preferably 15 min. After the third dehydration, the present invention preferably performs a fourth dehydration on the third dehydrated product in an 80% (v / v) ethanol aqueous solution to obtain a fourth dehydrated product. In the present invention, the fourth dehydration time is preferably 10-15 min, more preferably 15 min. After the fourth dehydration, the present invention preferably performs a fifth dehydration on the fourth dehydrated product in a 90% (v / v) ethanol aqueous solution to obtain a fifth dehydrated product. In the present invention, the fifth dehydration time is preferably 10-15 min, more preferably 15 min. After the fifth dehydration, the present invention preferably performs a sixth dehydration on the fifth dehydrated product in a 95% (v / v) ethanol aqueous solution to obtain a sixth dehydrated product. In the present invention, the sixth dehydration time is preferably 10-15 min, more preferably 15 min. After the sixth dehydration is completed, the present invention preferably performs a seventh dehydration on the sixth dehydrated product in anhydrous ethanol to obtain a seventh dehydrated product. In the present invention, the seventh dehydration time is preferably 10-15 minutes, more preferably 15 minutes. After the seventh dehydration is completed, the present invention preferably performs an eighth dehydration on the seventh dehydrated product in anhydrous ethanol to obtain an eighth dehydrated product. In the present invention, the eighth dehydration time is preferably 15-20 minutes, more preferably 20 minutes. In the present invention, the eighth dehydrated product is dehydrated plant tissue.

[0064] After obtaining the dehydrated plant tissue, the present invention preferably embeds the dehydrated plant tissue using Spurr embedding agent to obtain embedded tissue.

[0065] The embedding method described in this invention preferably employs a gradient permeation approach for gradual embedding. In this invention, the gradual embedding process preferably includes the following steps: immersing dehydrated plant tissue in acetone; the immersion time is preferably 20–30 min, more preferably 20 min. After immersion, the plant tissue soaked in acetone is preferably first embedded in a mixture of Spurr embedding agent and acetone at a volume ratio of 1:1 to obtain a first embedded product. In this invention, the temperature of the first embedding is preferably 22℃–28℃, more preferably 24℃; the first embedding time is preferably 1–1.5 h, more preferably 1 h. After the first embedding, the first embedded product is preferably second embedded in a mixture of Spurr embedding agent and acetone at a volume ratio of 3:1 to obtain a second embedded product. In this invention, the temperature of the second embedding is preferably 22℃–28℃, more preferably 24℃; the second embedding time is preferably 3–3.5 h, more preferably 3 h. After the second embedding is completed, the present invention preferably performs a third embedding in Spurr embedding medium to obtain plant tissue embedded blocks. In the present invention, the temperature of the third embedding is preferably 22℃~28℃, more preferably 24℃; the time of the third embedding is preferably 12~15h, more preferably 12h. After the third embedding is completed, the present invention obtains embedded tissue. The present invention does not have a special limitation on the amount of reagents used in the gradual embedding process, and conventional amounts in the art can be used.

[0066] After obtaining the embedded tissue, the present invention preferably polymerizes the embedded tissue to obtain a polymerized embedded block.

[0067] After obtaining the embedded tissue, the present invention preferably transfers the embedded tissue to Spurr embedding medium, and more preferably places two pieces of embedded tissue in the embedding medium in a "V" shape. The present invention does not have a special limitation on the amount of embedding medium used; conventional amounts in the art are sufficient. In the present invention, when embedding two pieces of tissue, the amount of embedding medium is preferably 150–250 μL, more preferably 200 μL. After obtaining the tissue coated with the embedding medium, the present invention preferably polymerizes the tissue coated with the embedding medium. The polymerization temperature is preferably 60°C–70°C, more preferably 70°C; the polymerization time is preferably 12–24 h, more preferably 12 h. After polymerization, the present invention obtains a polymerized embedded block. In the present invention, the process of polymerizing the embedded tissue to obtain the polymerized embedded block is a process in which the viscous Spurr embedding medium is transformed into a solid form, and the final polymerized embedded block is a solid embedded block. The polymerization time described in this invention should not be too long or too short to prevent the solid embedding block from becoming too hard or too soft. This invention uses 70°C for polymerization, which only requires 12 hours and can be completed overnight, greatly saving experimental operations. Furthermore, excessive polymerization time may cause the sample embedding block to become too hard and the sample to become brittle.

[0068] After obtaining the polymer-embedded block, the present invention preferably performs ultrathin sectioning on the polymer-embedded block to obtain ultrathin sections of plant tissue.

[0069] The sectioning described in this invention is preferably performed on a microtome. When performing ultrathin sectioning, the thickness of the section is preferably 70–90 nm, more preferably 70 nm. During sectioning, the plant tissue with the largest exposed area is preferably exposed. After sectioning, this invention obtains ultrathin sections of plant tissue.

[0070] After obtaining ultrathin sections of plant tissue, this invention performs immunochromatographic analysis on the ultrathin sections of plant tissue using colloidal gold electron microscopy.

[0071] In this invention, it is preferable to perform immunostaining on ultrathin sections of plant tissue before conducting immunochromatographic examination using colloidal gold electron microscopy. In this invention, the method for immunostaining ultrathin sections of plant tissue preferably includes the following steps:

[0072] Plant tissue ultrathin sections are eroded and sealed to obtain sealed ultrathin sections;

[0073] The blocked ultrathin sections were incubated with primary antibody to obtain primary antibody ultrathin sections;

[0074] The primary antibody ultrathin sections were incubated with gold-labeled secondary antibody to obtain gold-labeled secondary antibody ultrathin sections;

[0075] The ultrathin sections of the gold-labeled secondary antibody were stained to obtain ultrathin sections of plant tissue labeled with immunocolloidal gold.

[0076] Before etching the ultrathin sections of plant tissue, this invention preferably places the ultrathin sections of plant tissue on the front side of a nickel mesh and blots off the surface moisture with filter paper to obtain nickel-mesh ultrathin sections of plant tissue. This invention also preferably stores the nickel-mesh ultrathin sections of plant tissue in a mesh box for later use.

[0077] After obtaining ultrathin sections of nickel mesh plant tissue, the present invention preferably erodes and seals the ultrathin sections of nickel mesh plant tissue to obtain sealed ultrathin sections.

[0078] The etching process of this invention preferably includes: etching and incubating ultrathin sections of nickel-mesh plant tissue with an aqueous solution containing 10% hydrogen peroxide by volume. Preferably, the ultrathin sections of nickel-mesh plant tissue are placed upside down on a droplet containing 10% hydrogen peroxide by volume for etching and incubation. The etching and incubation temperature is preferably 20℃~28℃, more preferably 24℃; the etching and incubation time is preferably 10~15 min, more preferably 10 min. After etching and incubation, etched sections are obtained.

[0079] After obtaining the etched section, the present invention preferably absorbs the moisture from the surface of the etched section and then cleans the dried etched section. The cleaning is preferably performed using double-distilled water. The preferred cleaning method is to invert the dried etched section onto double-distilled water for cleaning. The cleaning is preferably performed three times; each cleaning session is preferably 5 minutes. The main purpose of cleaning in this invention is to remove residual hydrogen peroxide from the nickel mesh surface.

[0080] After cleaning, the present invention preferably uses filter paper to absorb the moisture on the surface of the sections before sealing them. The sealing process preferably includes sealing the ultrathin sections of plant tissue with a 1% (w / w) BSA aqueous solution. The present invention preferably places the cleaned and dried etched sections upside down on a 1% (w / w) BSA aqueous solution for sealing. The sealing temperature is preferably 20℃~28℃, more preferably 24℃; the sealing time is preferably 1~2 hours, more preferably 1.5 hours. After sealing, the present invention preferably uses filter paper to absorb the moisture from the sections, obtaining sealed ultrathin sections.

[0081] After obtaining the blocked ultrathin section, the present invention preferably incubates the blocked ultrathin section with a primary antibody to obtain a primary antibody ultrathin section.

[0082] The selection of the primary antibody in this invention is not particularly limited; it is sufficient to select an antibody that can specifically bind to the antigen to be tested and can be detected by immunohistochemistry, based on the properties of the antigen. In this invention, the primary antibody preferably includes Sar1 antibody or 1,3-β-glucan antibody.

[0083] In this invention, the primary antibody is preferably diluted before incubation. There is no specific limitation on the dilution factor of the primary antibody; dilution can be performed according to the antibody instructions. The primary antibody is preferably diluted using a 1% (w / w) BSA aqueous solution. The primary antibody incubation preferably includes: placing the blocked ultrathin section on the primary antibody dilution solution for incubation. Preferably, the blocked ultrathin section is mixed with the primary antibody dilution solution for incubation; more preferably, the blocked ultrathin section is inverted onto a droplet of the primary antibody dilution solution for incubation. The incubation temperature is preferably 20℃~28℃, more preferably 24℃; the incubation time is preferably 1~2 hours, more preferably 2 hours. After incubation, the sections are preferably blotted dry with filter paper and then washed. The washing is preferably done with double-distilled water. The washing preferably includes washing the sections inverted on double-distilled water. The washing is preferably performed three times; each washing session is preferably 5 minutes. The main purpose of cleaning the slides after primary antibody incubation in this invention is to ensure that there is no excess primary antibody residue on the slides. After cleaning, this invention preferably uses filter paper to absorb the moisture from the slides, obtaining ultrathin primary antibody slides.

[0084] After obtaining the primary antibody ultrathin section, the present invention preferably incubates the primary antibody ultrathin section with gold-labeled secondary antibody to obtain gold-labeled secondary antibody ultrathin section.

[0085] This invention places specific limitations on the selection of the gold-labeled secondary antibody during incubation. Any gold-labeled secondary antibody that matches the origin of the primary antibody and is conventionally available in the art is acceptable. In this invention, the gold-labeled secondary antibody is preferably a secondary antibody labeled with gold particles, and more preferably a secondary antibody labeled with gold particles of a fixed size. This invention does not place specific limitations on the size of the gold particles; any commercially available product conventional in the art is acceptable. In this invention, the preferred size of the gold particles is 10 nm. This invention preferably involves diluting the gold-labeled secondary antibody before incubation. In this invention, if the primary antibody 1,3-β-glucan antibody is mouse-derived, then a goat anti-mouse gold-labeled secondary antibody is selected; if the primary antibody Sar1 antibody is rabbit-derived, then a goat anti-rabbit gold-labeled secondary antibody is selected.

[0086] In this invention, the secondary antibody is preferably diluted before incubation. There is no particular limitation on the dilution factor of the secondary antibody; a conventional antibody dilution factor in the art can be used. Preferably, the secondary antibody is diluted using a 1% BSA aqueous solution. The secondary antibody incubation preferably includes: mixing the primary antibody ultrathin section with the gold-labeled secondary antibody dilution solution and incubating with the gold-labeled secondary antibody; more preferably, the primary antibody ultrathin section is placed on the gold-labeled secondary antibody dilution solution for incubation. Preferably, the primary antibody ultrathin section is inverted onto a droplet of gold-labeled secondary antibody dilution solution for incubation. The incubation temperature is preferably 20℃~28℃, more preferably 24℃; the incubation time is preferably 1~2 hours, more preferably 2 hours. After incubation, the sections are preferably blotted dry with filter paper and washed. Double-distilled water is preferably used for washing. In this invention, the cleaning process preferably involves inverting the slides onto double-distilled water for cleaning. The cleaning is preferably performed three times, with each cleaning session lasting approximately 5 minutes. Cleaning the slides after secondary antibody incubation is primarily to ensure that no excess secondary antibody residue remains on the slides. After cleaning, the slides are preferably blotted dry with filter paper to obtain ultrathin secondary antibody slides.

[0087] After obtaining the secondary antibody ultrathin section, the present invention preferably stains the secondary antibody ultrathin section to obtain immunogold-labeled plant tissue ultrathin section, which is then examined under an electron microscope.

[0088] The staining method of this invention preferably includes uranium acetate staining and lead citrate staining. After obtaining the secondary antibody ultrathin section, this invention preferably performs a first staining on the secondary antibody ultrathin section with uranium acetate; the first staining time is preferably 5-10 minutes, more preferably 10 minutes. After the first staining is completed, this invention preferably rinses the first-stained section to obtain a first-stained section. The rinsing method of this invention preferably includes rinsing with double-distilled water. This invention does not have a special limitation on the rinsing method, and any conventional rinsing method in the art can be used. After obtaining the first-stained section, this invention preferably performs a second staining on the first-stained section with lead citrate; the second staining time is preferably 5 minutes. This invention does not have a special limitation on the methods of uranium acetate staining and lead citrate staining, and any conventional staining method in the art can be used. After the second staining is completed, this invention preferably rinses the second-stained section to obtain a second-stained section. The rinsing method of this invention preferably includes rinsing with double-distilled water. This invention does not have a special limitation on the rinsing method, and any conventional rinsing method in the art can be used. After obtaining the second stained section, the present invention preferably bakes the second stained section; the baking time is preferably 8-10 minutes, more preferably 10 minutes. After baking, the present invention obtains an immunochromatographically labeled ultrathin section of plant tissue.

[0089] The ultrathin sections of plant tissue prepared by this invention with immunochromatographic colloidal gold labeling can be observed and photographed under a transmission electron microscope.

[0090] In the immunogold labeling method for plant tissue embedded slides provided by this invention, except for the initial material fixation and embedding which requires PBS buffer, subsequent steps (from the beginning of the immunostaining step, the entire cleaning process in this invention uses double-distilled water, while most staining steps using other embedding agents use PBS for cleaning) all use environmentally friendly and cost-effective double-distilled water. This reduces and minimizes the influence of PBS components on slide staining, greatly simplifies the experimental procedures, and has strong practicality. The blocking and antibody incubation steps in the immunogold labeling method for plant tissue embedded slides do not need to be performed at 37°C; they can be performed at room temperature, reducing the adverse effects of temperature on the slides (incubation at 37°C may cause buffer components to deposit on the slides, creating impurities that affect slide observation), and also simplifies the experimental procedures. This invention's method is simple and easy to implement, produces less contamination in electron microscopy results, and has a high success rate. The method for immunogold labeling of plant tissue embedded slides provided by this invention avoids the use of complex low-temperature polymerized embedding agents, and has the advantages of good contrast and high contrast. Moreover, the prepared slides can be used for various experiments such as immunogold electron microscopy and ultrastructure observation. The operation is convenient, quick and efficient, and can be widely promoted and applied.

[0091] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0092] Example 1

[0093] Immunogold electron microscopy of Spurr resin ultrathin sections of K326 leaves.

[0094] The immunogold labeling method for plant tissue embedded slides is implemented as follows:

[0095] 1) Sampling and fixation: Fresh leaf tissue of tobacco K326 cultured in the experimental greenhouse was collected. The leaves were cut into pieces no larger than 1×2 mm on a clean glass slide using a new Gillette blade. 2 Four small pieces of the leaf were placed in a clean 2.0 mL centrifuge tube, and 600 μL of 2.5% glutaraldehyde was added immediately for pre-fixation. The centrifuge tube containing the leaf and the 2.5% glutaraldehyde aqueous solution was placed in a vacuum filtration device and vacuumed for 10 min to allow the leaf to adhere to the bottom of the centrifuge tube. The fixed sample was placed in a refrigerator at 4°C for 24 h, and then rinsed with 0.1 M PBS for 15 min, repeated 3 times. Subsequently, the sample was post-fixed with 250 μL of 1 wt.% osmium tetroxide aqueous solution at 24°C for 2 h.

[0096] 2) Dehydration and Embedding: After rinsing three times with 0.1M PBS (15 min each time), the sample was sequentially immersed in 30%, 50%, 70%, 80%, 90%, 95%, and 100% anhydrous ethanol for 15 min each, followed by dehydration with 100% anhydrous ethanol for 20 min, and then soaked in acetone for 20 min. The dehydrated sample was then subjected to gradient permeation with different ratios of embedding agents. First, it was treated in a 1:1 mixture of Spurr embedding agent and acetone at 24°C for 1 h to obtain the first embedded plant tissue. After obtaining the first embedded plant tissue, it was treated in a 3:1 mixture of Spurr embedding agent and acetone at 24°C for 3 h to obtain the second embedded plant tissue. Finally, the second embedded plant tissue was treated in pure Spurr embedding agent at 24°C for 12 h to ensure sufficient permeation of the sample.

[0097] 3) Resin polymerization: Add 150 μL of Spurr embedding medium to a 0.5 mL centrifuge tube. Use a toothpick to gently pick up the sample (of similar size, shape, and volume, without damage) that has been infiltrated overnight in step 2) onto clean filter paper. Then use a toothpick to pick up the sample into 150 μL of Spurr embedding medium, so that the two leaf pieces are placed in the embedding medium in a "V" shape. Then place the 0.5 mL centrifuge tube containing the sample and embedding medium into the polymerization chamber and polymerize at 70 °C for 12 h. After polymerization, remove it for use.

[0098] 4) Ultrathin sectioning: First, remove the polymerized embedded block from the centrifuge tube, fix the embedded block on the microtome to expose the largest area of ​​tissue, correct the positioning, and then perform ultrathin sectioning with a section thickness of 70 nm. Place the sectioned slides on a nickel mesh, blot dry with filter paper, and obtain nickel mesh plant tissue ultrathin sections. Place the nickel mesh plant tissue ultrathin sections in a mesh box for later use.

[0099] 5) Etching and Sealing: Prepare a 10% (v / v) hydrogen peroxide aqueous solution droplet (referred to as 10% H2O2 droplet). Invert the nickel mesh plant tissue ultrathin section prepared in step 4) onto the 10% H2O2 droplet and incubate at room temperature for 10 min to erode the section. Then, blot the section dry with filter paper and invert it onto double-distilled water 3 times, 5 min each time. Prepare a 1% BSA blocking solution (referred to as 1% BSA) using double-distilled water. Blot the section dry with filter paper and invert it onto the 1% BSA solution, sealing at 24℃ for 2 h.

[0100] 6) Primary antibody incubation: Dilute primary antibody Sar1 with 1% BSA blocking buffer (dilution ratio is 1:50), blot the slide dry with filter paper, and then invert it onto the diluted primary antibody droplet. Incubate at 24℃ for 2 hours.

[0101] 7) Secondary antibody incubation: Dilute the colloidal gold-labeled goat anti-rabbit secondary antibody with 1% BSA (dilution ratio 1:50), then blot the slide dry with filter paper and invert it onto double-distilled water 3 times, 5 min each time. Finally, blot the slide dry with filter paper and invert it onto the diluted secondary antibody droplet, incubate at 24℃ for 2 h;

[0102] 8) Lead-uranium double staining: After blotting the slides with filter paper, invert them over double-distilled water three times, 5 minutes each time. First, stain with uranium acetate for 10 minutes and rinse with double-distilled water, then stain with lead citrate for 5 minutes and rinse with double-distilled water. Finally, place the slides under a baking lamp and bake for 10 minutes to obtain immunochromatographically labeled ultrathin sections of plant tissue.

[0103] After obtaining the immunogold-labeled ultrathin sections of plant tissue, the sections can be observed and photographed under a transmission electron microscope using an appropriate voltage and current.

[0104] Observe the photographic results as follows Figure 1 As shown. Figure 1 The white arrows indicate virus particles; the yellow arrows indicate gold particles; and 'w' represents the cell wall.

[0105] This embodiment uses K326 cells cultivated in a laboratory greenhouse as experimental material. The method of this invention was employed for immunogold electron microscopy of Sar1 antibodies, yielding clear images with high contrast, clear viral morphology and cellular ultrastructure, and clearly labeled gold particles. Sar1 antibodies can recognize the Sar1 protein, which is a protein localized to the endoplasmic reticulum. Therefore, the gold particles detected by Sar1 antibodies will deposit near the endoplasmic reticulum membrane. Figure 1 The area covered by the gold particles had a section of incomplete endoplasmic reticulum membrane. No gold particles aggregated in other locations within the cell, indicating that the test results were accurate and no false-positive gold particle deposition occurred.

[0106] Comparative Example 1

[0107] Immunogold electron microscopy of Spurr resin ultrathin sections of K326 leaves.

[0108] The experimental steps of the immunogold labeling method for plant tissue embedded slides are the same as in Example 1, except that primary antibody incubation is not performed.

[0109] Observe the photographic results as follows Figure 2 As shown. Figure 2 The white arrows in the image indicate virus particles.

[0110] Figure 2 As a negative control for the experiment, almost no gold particle deposition was observed in the image.

[0111] Depend on Figure 1and Figure 2 It was found that, in the presence of the primary antibody, gold particles accurately detected the presence of the antigen and aggregated, while almost no gold particles were observed in the negative control without the primary antibody. This indicates that the experimental procedure of the present invention is accurate and did not cause false positives in gold particles or impurities.

[0112] Example 2

[0113] Immunocolloid gold electron microscopy of Spurr resin ultrathin sections of tobacco leaves

[0114] In this embodiment, *Nicotiana benthamiana* cultivated in the laboratory was used as the experimental material, and the method of this invention was employed to detect the β-glucan target protein using immunochromatographic gold electron microscopy.

[0115] The implementation process is basically the same as in Implementation Example 1, with only the following differences:

[0116] 1) Primary antibody incubation: Dilute the primary antibody β-glucan (1,3-β-glucan antibody) with 1% BSA blocking buffer (dilution ratio is 1:100), blot the slide dry with filter paper, invert it onto the diluted primary antibody droplet, and incubate at room temperature for 2 hours;

[0117] 2) Secondary antibody incubation: Dilute the colloidal gold-labeled goat anti-mouse secondary antibody with 1% BSA (dilution ratio 1:100), then blot the slide dry with filter paper and invert it onto double-distilled water 3 times, 5 min each time. Finally, blot the slide dry with filter paper and invert it onto the diluted secondary antibody droplet, and incubate at room temperature for 2 h.

[0118] Observe the photographic results as follows Figure 3 As shown.

[0119] Comparative Example 2

[0120] Immunocolloid gold electron microscopy of Spurr resin ultrathin sections of tobacco leaves

[0121] The experimental steps of the immunogold labeling method for plant tissue embedded slides are the same as in Example 2, except that primary antibody incubation was not performed.

[0122] Observe the photographic results as follows Figure 4 As shown.

[0123] Depend on Figure 3 and Figure 4 It was found that β-glucan is mainly found in plasmodesmata of plant cell walls. In the presence of β-glucan primary antibody, gold particles were observed deposited at the plasmodesmata, while in the negative control without primary antibody, only a few gold particles were observed at the plasmodesmata. This indicates that the experimental method of this invention is accurate and did not cause false positives or the appearance of gold particles or impurities.

[0124] Comparative Example 3

[0125] Immunogold-labeled ultrathin sections of plant tissue were prepared using K4M low-temperature embedding medium.

[0126] Experimental immunogold electron microscopy detection method for K4M embedding agent:

[0127] (1) Material preparation and fixation: Cut the Benedict tobacco leaves into 1×3mm pieces 2 The small pieces were then placed in a 4% paraformaldehyde fixative containing 0.1% glutaraldehyde and fixed overnight in a refrigerator at 4°C, ensuring that the leaves were submerged in the fixative solution.

[0128] (2) Washing: Use a pipette to remove as much fixative as possible, and wash the leaves 5 times with 0.1M PBS for 8 minutes each time. This step is performed in a 4°C refrigerator.

[0129] (3) Dehydration: The leaves were placed in 30% (30 min), 50% (30 min), 70% (1 h), 90% (1 h), 100% (1 h), 100% (1 h), and 100% (1 h) of ethanol for gradient dehydration. The dehydration time is shown in parentheses. Dehydration was carried out at 4℃.

[0130] (4) Washing: Take out the dehydrated leaves and wash them 5 times in 0.1M PBS for 10 minutes each time. This step is carried out in a 4℃ refrigerator.

[0131] (5) Prepare K4M embedding agent (S2643, Lowicryl) according to the instructions.

[0132] (6) Gradient permeation: After washing, the leaves were placed in a mixture of ethanol and K4M embedding agent at a volume ratio of 3:1 at 4°C for 1 hour. Then, the leaves were placed in a mixture of ethanol and K4M embedding agent at a volume ratio of 1:1 at 4°C for 3 hours. Finally, the leaves were placed in pure K4M embedding agent overnight at 4°C (12 hours) for gradient permeation. Finally, 3-5 leaves were placed vertically into 0.5 mL EP tubes containing 300 μL of pure embedding agent.

[0133] (7) Ultraviolet polymerization: Place the EP tube containing the permeated leaf from step (6) into an ultraviolet polymerization box, and then place the ultraviolet polymerization box containing the EP tube into a -20°C refrigerator for polymerization for 72 hours. After that, take out the polymerization box and place it at room temperature (24°C) for polymerization for 48 hours.

[0134] (8) Ultrathin sectioning and slide retrieval: The embedded block was sectioned at 90 nm on an ultrathin slicer and then retrieved using a nickel mesh.

[0135] (9) Washing: Invert the nickel mesh containing the slices onto 0.1M PBS and wash 3 times for 5 min each time. Then wash in double-distilled water in the same way (wash 3 times for 5 min each time).

[0136] (10) Fixation: Invert the nickel mesh into 1% BSA (containing 10% cold-water fish gelatin) and incubate at room temperature (24℃) for 30 min.

[0137] (11) Primary antibody: Invert the nickel mesh into the primary antibody 1,3-β-glucan antibody diluted with blocking solution (dilution ratio of 1:100) and incubate at 37°C for 2 hours.

[0138] (12) Washing: The nickel mesh containing the slices was inverted and washed three times with 0.1M PBS for 5 hours each time. Then it was washed in double-distilled water in the same way (washed three times with double-distilled water for 5 minutes each time).

[0139] (13) Secondary antibody: The nickel mesh was placed upside down in the goat anti-mouse (1:100) secondary antibody labeled with gold particles diluted with blocking solution and incubated at 37°C for 1 hour.

[0140] (14) Washing: Invert the nickel mesh containing the slices onto 0.1M PBS and wash 3 times for 5 min each time. Then wash in double-distilled water in the same way (wash 3 times for 5 min each time).

[0141] (15) Double staining: After slightly drying the prepared slides, stain them in 2% uranium acetate for 15 minutes, rinse with 0.1M PBS for 5 minutes, stain them in lead citrate for 7 minutes, rinse with 0.1M PBS for 5 minutes, and then bake them dry.

[0142] (16) Observation: Observe and photograph under a transmission electron microscope.

[0143] Electron microscopy observations of ultrathin sections of plant tissue prepared using K4M low-temperature embedding medium and labeled with colloidal gold are as follows: Figure 5 As shown.

[0144] The polymerization time of low-temperature embedding agents is relatively long. Results of low-temperature embedding agent treatment: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Figure 5 It can be seen that the contrast of organelles in the K4M low-temperature embedding agent results is not strong, and the cell wall has low contrast and is not easy to observe under electron microscopy.

[0145] Comparative Example 4

[0146] Immunogold-labeled ultrathin sections of plant tissue were prepared using 812 embedding medium.

[0147] (1) Material preparation and fixing: Cut the Benedict tobacco leaves into 1×3mm pieces. 2The rectangular blocks were then fixed in 2.5% glutaraldehyde-PBS aqueous solution and fixed overnight at 4°C (fixation time was 12h).

[0148] (2) Washing: Discard the fixative and wash 5 times with 0.1M pre-cooled PBS for 8 minutes each time. This step is performed in a 4°C refrigerator.

[0149] (3) Post-fixation: The leaf was placed in a 1% osmium tetroxide aqueous solution and post-fixed at 4°C for 2 hours.

[0150] (4) Washing: After removing the leaves, wash them 5 times with 0.1M PBS for 10 minutes each time. This step is carried out in a 4℃ refrigerator.

[0151] (5) Dehydration: The leaves were placed in 30% (30 min), 50% (30 min), 70% (1 h), 80% (1 h), 90% (1 h), 95% (1 h), 100% (1 h), and 100% (1 h) of ethanol for gradient dehydration. The dehydration time is shown in parentheses. Dehydration was carried out at 4°C.

[0152] (6) Excessive use of acetone: After dehydration, the leaves were placed in a mixture of anhydrous ethanol and acetone in a volume ratio of 3:1 for 0.5 h, then in a mixture of anhydrous ethanol and acetone in a volume ratio of 1:1 for 0.5 h, and finally in pure acetone for 1 h.

[0153] (7) Gradient permeation embedding: The leaf treated in step (6) was placed in a mixture of acetone and 812 embedding agent at a volume ratio of 3:1 for 4 hours, then in a mixture of acetone and 812 embedding agent at a volume ratio of 1:1 overnight (12 hours), then in a mixture of acetone and 812 embedding agent at a volume ratio of 1:3 for 4 hours, and finally infiltrated with pure 812 embedding agent for 8 hours. Finally, the leaf was placed in an embedding plate containing pure 812 embedding agent and placed at 37°C overnight.

[0154] (8) High-temperature polymerization: The well-permeated embedding plate was placed in a 60℃ oven for polymerization for 48 hours.

[0155] (9) Washing: Invert the nickel mesh containing the slices onto 0.1M PBS and wash 3 times for 5 min each time. Then wash in double-distilled water in the same way (wash 3 times for 5 min each time).

[0156] (10) Fixation: Invert the nickel mesh into 1% BSA (containing 10% cold-water fish gelatin) and incubate at room temperature for 30 min.

[0157] (11) Primary antibody: Invert the nickel mesh into the primary antibody 1,3-β-glucan antibody diluted with blocking solution (dilution ratio of 1:100) and incubate at 37°C for 2 hours.

[0158] (12) Washing: Invert the nickel mesh containing the slices onto 0.1M PBS and wash 3 times for 5 min each time. Then wash in double-distilled water in the same way (wash 3 times for 5 min each time).

[0159] (13) Secondary antibody: The nickel mesh was placed upside down in the secondary antibody labeled with gold particles (dilution ratio of 1:100) diluted with blocking solution and incubated at 37°C for 1 hour.

[0160] (14) Washing: Invert the nickel mesh containing the slices onto 0.1M PBS and wash 3 times for 5 min each time. Then wash in double-distilled water in the same way (wash 3 times for 5 min each time).

[0161] (15) Double staining: After slightly drying the prepared slides, first stain them in 2% uranium acetate for 15 min, rinse them with 0.1M PBS for 5 min, then stain them in lead citrate for 7 min, rinse them with 0.1M PBS for 5 min, and then bake them dry.

[0162] (16) Observation: Observe and photograph under a transmission electron microscope.

[0163] Electron microscopy observations of ultrathin sections of plant tissue prepared using 812 embedding medium and labeled with colloidal gold are as follows: Figure 6 As shown.

[0164] Depend on Figure 6 It can be seen that the organelle contrast in the 812 embedding medium results is acceptable, but non-specific deposition of gold particles is prone to occur in colloidal gold detection. Non-specific deposition is equivalent to false positives. From Figure 6 As can be seen, the deposition of gold particles is irregular and almost scattered. Even in blank vacuoles where no organelles are present, gold particles can be observed to be scattered.

[0165] Comparative Example 5

[0166] Immunogold electron microscopy technique for ultrathin sections of K326 leaf tissues in Spurr resin.

[0167] The experimental steps of the immunogold labeling method for plant tissue embedded slides are the same as in Example 1, except that the washing steps are performed by washing with 0.1M PBS and then washing with double-distilled water.

[0168] Observe the photographic results as follows Figure 7 As shown. Figure 7 The white arrows in the image indicate impurity deposits.

[0169] Depend on Figure 7 It can be concluded that, compared with washing with only double-distilled water, washing with 0.1M PBS buffer may result in impurity deposition, affecting the observation and aesthetics of the slides.

[0170] Comparative Example 6

[0171] Immunogold electron microscopy technique for ultrathin sections of K326 leaf tissues in Spurr resin.

[0172] The experimental steps of the immunogold labeling method for plant tissue embedded slides are the same as in Example 1, except that the primary and secondary antibodies are incubated at 37°C.

[0173] Observe the photographic results as follows Figure 8 As shown. Figure 8 The white arrow in the image indicates the impurity deposits formed at 37°C.

[0174] Depend on Figure 8 Therefore, incubation at 37°C will cause buffer substances to deposit on the slide, affecting the observation results.

[0175] In summary, the immunogold labeling method for plant tissue embedded slides provided by this invention uses Spurr embedding agent to embed plant tissue, followed by immunogold labeling for detection. This method yields detection results with clear virus morphology and cell ultrastructure, high gold particle contrast, and no false positives.

[0176] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for detecting Sar 1 using immunocolloidal gold electron microscopy on ultrathin sections of Spurr-embedded plant tissue samples, characterized in that, Includes the following steps: Plant tissues were embedded using Spurr embedding medium to obtain ultrathin sections of plant tissue; The method for preparing ultrathin sections of plant tissue includes the following steps: Plant tissues are fixed to obtain fixed tissues; the fixation includes a first fixation and a second fixation; the first fixation is performed using a 2.5% (v / v) glutaraldehyde aqueous solution for 24-48 hours at a temperature of 4-8°C; the second fixation is performed using a 1% (v / v) osmium tetroxide aqueous solution for 1.5-2 hours at a temperature of 20-28°C. After dehydration, the fixed tissue was embedded using Spurr embedding medium to obtain embedded tissue. The embedding process included: immersing the dehydrated plant tissue in acetone to obtain acetone-soaked plant tissue for 20–30 minutes; first embedding the acetone-soaked plant tissue in a 1:1 volume ratio of Spurr embedding medium to acetone to obtain a first embedded product for 1–1.5 hours; second embedding the first embedded product in a 3:1 volume ratio of Spurr embedding medium to acetone to obtain a second embedded product for 3–3.5 hours; and third embedding the second embedded product in Spurr embedding medium to obtain a plant tissue embedding block for 12–15 hours. The embedding temperature was 22–28°C. The embedded tissue was polymerized in Spurr embedding agent to obtain polymerized embedded blocks; the polymerization temperature was 60℃~70℃ and the polymerization time was 12~24h. The polymer-embedded blocks were subjected to ultrathin sectioning to obtain ultrathin sections of plant tissue; Immunocolloid gold electron microscopy was performed on ultrathin sections of plant tissue. After obtaining ultrathin sections of plant tissue, the method for immunogold electron microscopy detection includes the following steps: Plant tissue ultrathin sections are eroded and sealed to obtain sealed ultrathin sections; The blocked ultrathin sections were incubated with primary antibody and washed three times with double-distilled water to obtain primary antibody ultrathin sections. The primary antibody ultrathin sections were incubated with gold-labeled secondary antibody and washed three times with double-distilled water to obtain gold-labeled secondary antibody ultrathin sections. The ultrathin sections of gold-labeled secondary antibody were stained and washed three times with double-distilled water to obtain ultrathin sections of plant tissue labeled with immunocolloidal gold, which were then examined under an electron microscope. The incubation temperature for the primary antibody is 20℃~28℃; the incubation time for the primary antibody is 1~2h; the incubation temperature for the secondary antibody is 20℃~28℃; the incubation time for the secondary antibody is 1~2h.

2. The method according to claim 1, characterized in that, The sealing process includes: sealing ultrathin sections of plant tissue with a 1% (w / w) BSA aqueous solution; The temperature of the sealing treatment is 20℃~28℃; the sealing treatment time is 1~2h.

3. The method according to claim 1, characterized in that, The dehydration process involves gradually dehydrating with ethanol aqueous solutions of different concentrations, followed by final dehydration with anhydrous ethanol to obtain dehydrated plant tissue. The ethanol aqueous solutions of different concentrations include, in sequence, ethanol aqueous solutions with a volume percentage of 30%, 50%, 70%, 80%, 90%, and 95%. The anhydrous ethanol dehydration treatment was performed twice.

4. The method according to claim 1, characterized in that, Staining methods include uranium acetate staining and lead citrate staining.

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