Fixing agent for ultrathin section electron microscope technology as well as preparation method and application of fixing agent

Abstract

The invention relates to a fixing agent for an ultrathin section electron microscope technology and a preparation method and application of the fixing agent. The fixing agent comprises tannic acid, potassium permanganate and uranyl acetate. The fixing agent comprises tannic acid, potassium permanganate and uranyl acetate, the three components are lower in toxicity compared with osmic acid, after a sample is fixed in an experiment, the sample is clear in structural form and easy to distinguish, the fixing effect of the sample is equivalent to that of the osmic acid, and the safety risk of using the osmic acid can be reduced by adopting the fixing agent; and meanwhile, the application of the ultrathin section electron microscope technology can be further expanded.

Description

Technical Field

[0001] The invention relates to the technical field of transmission electron microscope sample preparation, in particular to a fixing agent used in ultrathin section electron microscope technology and a preparation method and application thereof. Background Art

[0002] Electron microscopy is an important tool for studying the ultrastructure and function of cells and tissues, and is widely used in the biomedical field. In addition, electron microscopy also plays an important role in virology research, especially in the identification of virus particles, morphological research and virus detection. Ultrathin section transmission electron microscopy is the most direct and effective way to detect the presence of virus particles. Whether from the perspective of virus morphology or virus genesis, clear and accurate results can be obtained through ultrathin section electron microscopy.

[0003] Fixation is one of the key steps in sample preparation for ultrathin section electron microscopy. The purpose of fixation is to preserve the structure of the tissue as close to the state of life as possible. At present, the commonly used fixation method in ultrathin section electron microscopy is double fixation with glutaraldehyde and osmium acid. The fixative osmium acid is highly toxic. Contact or inhalation can cause eye diseases and severe conjunctival inflammation, and can lead to headaches, respiratory inflammation, eye diseases, bronchial diseases, pneumonia, etc. Therefore, there is a great safety risk in its use. Osmium acid is also one of the highly toxic chemicals that are under important control. In order to avoid the occurrence of safety incidents caused by improper use, there are strict requirements for its use: not only the user unit is required to have comprehensive qualifications, including purchase permits, storage places, and account system entries, but also the users must participate in training and obtain officially authorized qualification certificates, and conduct regular audits every year, which greatly increases the management cost. It can be seen that osmium acid is difficult to obtain and has a high safety risk when used, which greatly increases the threshold of ultrathin section electron microscopy technology and limits the application of ultrathin section electron microscopy technology.

[0004] In order to solve the above technical problems, the present invention discloses a reagent scheme, which can replace the highly toxic osmium acid as a new fixative to reduce the safety risk of using osmium acid, and can further expand the application of ultrathin section electron microscopy technology. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a fixative for ultrathin section electron microscopy technology and its preparation method and application. The fixative in the present invention includes tannic acid, potassium permanganate and uranyl acetate. The above three components are less toxic than osmic acid. After the sample is fixed in the experiment, the sample structure and morphology are clear and easy to distinguish, and the fixation effect is equivalent to that of osmic acid. The use of the above fixative can reduce the safety risk of using osmic acid, and at the same time can further expand the application of ultrathin section electron microscopy technology.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a fixative for use in ultrathin section electron microscopy technology, wherein the fixative comprises tannic acid, potassium permanganate and uranyl acetate.

[0008] The present invention creatively combines tannic acid, potassium permanganate and uranyl acetate to obtain a good fixation effect, and the contrast of the fixed sample is moderate and the structure is clearly discernible. Tannic acid is an organic compound and is mainly used as a mordant in the present invention; potassium permanganate is a strong oxidant and is mainly used as a penetrant in the present invention; and uranyl acetate is a commonly used dye.

[0009] Preferably, the fixative is divided into a cell fixative and a virus fixative according to different treatment objects.

[0010] In the present invention, due to the differences in morphology, structure and size of cells and viruses, the concentration ranges of the components in the fixative also vary to a certain extent.

[0011] Preferably, the concentration of tannic acid in the cell fixative is 3.33-13.33 mg / mL, for example, it can be 3.33 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL or 13.33 mg / mL, etc.

[0012] Preferably, the concentration of potassium permanganate in the cell fixative is 1.80-3.64 mg / mL, for example, 1.80 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL or 3.64 mg / mL.

[0013] Preferably, the concentration of uranyl acetate in the cell fixative is 1.82-10 mg / mL, for example, 1.82 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL or 10 mg / mL.

[0014] Preferably, the concentration of tannic acid in the virus fixative is 2.86-13.33 mg / mL, for example, it can be 2.86 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL or 13.33 mg / mL, etc.

[0015] Preferably, the concentration of potassium permanganate in the virus fixative is 1.71-3.64 mg / mL, for example, 1.71 mg / mL, 2 mg / mL, 3 mg / mL or 3.64 mg / mL.

[0016] Preferably, the concentration of uranyl acetate in the virus fixative is 1.82-14.29 mg / mL, for example, it can be 1.82 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL or 14.29 mg / mL, etc.

[0017] In the present invention, a better fixation effect can be obtained within the above range. Outside this range, too low a value will lead to poor fixation effect and difficulty in subsequent observation; too high a value will damage the sample and cause distortion of the observation result.

[0018] In a second aspect, the present invention provides a method for preparing a fixative for use in ultrathin section electron microscopy technology according to the first aspect, the preparation method comprising dissolving tannic acid, uranyl acetate and potassium permanganate respectively and then mixing the three solutions.

[0019] Preferably, the solvent of the tannic acid comprises PBS buffer.

[0020] Preferably, the concentration of the tannic acid after dissolution is 0.02-0.06 g / mL, for example, it can be 0.02 g / mL, 0.03 g / mL, 0.04 g / mL, 0.05 g / mL or 0.06 g / mL.

[0021] Preferably, the solvent of the uranyl acetate comprises water.

[0022] Preferably, the concentration of the dissolved uranyl acetate is 0.02-0.05 g / mL, for example, 0.02 g / mL, 0.03 g / mL, 0.04 g / mL or 0.05 g / mL.

[0023] Preferably, the solvent for potassium permanganate comprises water.

[0024] Preferably, the concentration of the potassium permanganate after dissolution is 3-5 mg / mL, for example, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL or 5 mg / mL.

[0025] Preferably, the volume ratio of tannic acid, potassium permanganate and uranyl acetate in the cell fixative is 1:(3-4):(0.5-1.0). The (3-4) may be, for example, 3, 3.5 or 4. The (0.5-1.0) may be, for example, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0.

[0026] Preferably, the volume ratio of tannic acid, potassium permanganate and uranyl acetate in the virus fixative is 1:(3-4):(0.5-2.0). The (3-4) may be, for example, 3, 3.5 or 4. The (0.5-2.0) may be, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0.

[0027] The present invention finds that within this ratio range, the effects of the above components can be maximized, the sample can be effectively fixed, and the accuracy of detection can be improved.

[0028] In a third aspect, the present invention provides a detection method using an ultrathin section electron microscope, the detection method comprising sample pretreatment, fixing the sample using the fixative used in the ultrathin section electron microscope technology described in the first aspect, washing, dehydration, penetration, embedding and polymerization, and then ultrathin sectioning, staining the sections and then conducting detection.

[0029] Preferably, the sample comprises a cell sample and / or a virus sample.

[0030] Preferably, the cell sample pretreatment step comprises fixing the cell sample with glutaraldehyde, collecting cell clusters, embedding them into blocks with agar, and then washing them.

[0031] Preferably, the concentration of glutaraldehyde in the cell sample pretreatment is 2-5%, for example, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%.

[0032] Preferably, the washing solution comprises PBS buffer.

[0033] Preferably, the virus sample pretreatment step comprises collecting the precipitate after centrifuging the sample, making the precipitate into an agar block and then fixing it with glutaraldehyde.

[0034] Preferably, the centrifugal time is 1-2h, and the centrifugal speed is 80000-120000g, and the 1-2h can be, for example, 1h, 1.2h, 1.4h, 1.6h, 1.8h or 2h, etc. The 80000-120000g can be, for example, 80000g, 85000g, 90000g, 85000g, 100000g, 105000g, 110000g, 115000g or 120000g, etc.

[0035] Preferably, the concentration of glutaraldehyde in the virus sample pretreatment is 2-5%, for example, 2%, 3%, 4% or 5%.

[0036] Preferably, the volume of the fixative added to the fixed sample is 0.3-1.0 mL, for example, it can be 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL or 1.0 mL.

[0037] Preferably, the fixed time is 0.5-2h, for example, it can be 0.5h, 0.6h, 0.8h, 1.0h, 1.2h, 1.4h, 1.6h, 1.8h or 2h.

[0038] Preferably, the cleaning agent comprises deionized water.

[0039] Preferably, the dehydration and infiltration step comprises: dehydration treatment with gradient ethanol, followed by acetone treatment, and then adding gradient mixed solution for further treatment, and finally infiltration with pure resin.

[0040] Preferably, the concentration of the gradient ethanol in the virus sample ranges from 50% to 100%, for example, it may be 50%, 60%, 70%, 80%, 90% or 100%.

[0041] Preferably, in the gradient ethanol dehydration treatment of the virus sample, the dehydration time of each gradient ethanol is 10-20 min, for example, it can be 10 min, 15 min or 20 min.

[0042] Preferably, the concentration of the gradient ethanol in the cell sample is in the range of 30-50%, for example, 30%, 35%, 40%, 45% or 50%.

[0043] Preferably, in the gradient ethanol dehydration treatment of the cell sample, the dehydration time of each gradient ethanol is 10-20 min, for example, 10 min, 15 min or 20 min.

[0044] Preferably, the cell sample undergoes a second dehydration treatment and a third dehydration treatment after the dehydration treatment.

[0045] Preferably, the reagent for the second dehydration treatment is 60-80% ethanol, for example, 60%, 65%, 70%, 75% or 80%.

[0046] Preferably, the second dehydration treatment time is 6-12 h, for example, it can be 6 h, 7 h, 8 h, 9 h, 10 h, 11 h or 12 h.

[0047] Preferably, the reagent for the third dehydration treatment is 90-100% gradient ethanol, for example, 90%, 95% or 100%.

[0048] Preferably, in the third dehydration treatment, the dehydration time of each gradient ethanol is 10-20 min, for example, it can be 10 min, 12 min, 14 min, 16 min, 18 min or 20 min.

[0049] Preferably, the acetone treatment time is 10-20 min, for example, it can be 10 min, 12 min, 14 min, 16 min, 18 min or 20 min.

[0050] Preferably, the gradient mixed solution comprises acetone and resin.

[0051] Preferably, the volume ratio of acetone to resin in the gradient mixed solution is (1-2):(1-2). The (1-2) may be, for example, 1, 1.2, 1.4, 1.6, 1.8 or 2.

[0052] Preferably, the re-treatment time is 1-2 hours, for example, 1 hour, 1.5 hours or 2 hours.

[0053] Preferably, the number of re-treatments is 1-3 times, for example, 1 time, 2 times or 3 times.

[0054] Preferably, the penetration time is 6-12 h, for example, it can be 6 h, 7 h, 8 h, 9 h, 10 h, 11 h or 12 h.

[0055] Preferably, the temperature of the embedding polymerization is 45-60°C and the time is 6-12h. The 45-60°C may be, for example, 45°C, 46°C, 48°C, 50°C, 52°C, 54°C, 56°C, 58°C or 60°C. The 6-12h may be, for example, 6h, 7h, 8h, 9h, 10h, 11h or 12h.

[0056] Preferably, the staining step comprises staining with uranyl acetate and then staining with lead citrate.

[0057] Preferably, the concentration of uranyl acetate is 0.02-0.05 mg / mL, for example, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL or 0.05 mg / mL.

[0058] Preferably, the uranyl acetate staining time is 15-20 min, for example, it can be 15 min, 16 min, 17 min, 18 min, 19 min or 20 min.

[0059] Preferably, the concentration of the lead citrate is 0.02-0.05 mg / mL, for example, it can be 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL or 0.05 mg / mL.

[0060] Preferably, the dyeing time of the lead citrate is 10-15 min, for example, it can be 10 min, 11 min, 12 min, 13 min, 14 min or 15 min.

[0061] In a fourth aspect, the present invention provides a fixative for ultrathin section electron microscopy according to the first aspect or a detection method using an ultrathin section electron microscope according to the third aspect in ultrathin section electron microscopy detection.

[0062] Compared with the prior art, the present invention has at least the following beneficial effects:

[0063] 1. The present invention creatively combines tannic acid, potassium permanganate and uranyl acetate to form a new type of fixative. After the fixative is used to fix the sample in the experiment, the sample structure is clear and easy to distinguish. And the fixed sample has a good fixation effect whether it is a cell or a virus.

[0064] 2. The fixative of the present invention is less toxic than the osmium acid fixative commonly used on the market. The use of the fixative of the present invention can reduce the safety risk of using osmium acid and further expand the application of ultrathin section electron microscopy technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 This is a diagram showing the cell fixation effect of Application Example 1.

[0066] Figure 2 This is a diagram showing the cell fixation effect of comparative application example 1.

[0067] Figure 3 This is a diagram showing the cell fixation effect of Application Example 2.

[0068] Figure 4 This is a diagram showing the virus fixation effect of Application Example 3.

[0069] Figure 5This is a diagram showing the virus fixation effect of Application Example 4.

[0070] Figure 6 This is a diagram showing the virus fixation effect of comparative application example 2. DETAILED DESCRIPTION

[0071] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0072] Example 1

[0073] This example prepares the fixative for ultrathin section electron microscopy

[0074] Prepare cell fixative: Weigh 0.4g tannic acid and add 10mL PBS buffer to prepare a 0.04g / mL tannic acid solution. Add 0.6g uranyl acetate to 20mL water to prepare a 0.03g / mL uranyl acetate solution. Weigh 0.03g potassium permanganate and add 10mL water to prepare a 3mg / mL potassium permanganate solution.

[0075] The prepared tannic acid solution, potassium permanganate solution and uranyl acetate were mixed in a volume ratio of 1:4:0.5 to prepare a fixative, wherein the concentration of tannic acid after mixing was 7.27 mg / mL, the concentration of uranyl acetate was 2.73 mg / mL, and the concentration of potassium permanganate was 2.18 mg / mL.

[0076] Example 2

[0077] This example prepares the fixative for ultrathin section electron microscopy

[0078] Prepare cell fixative: Weigh 0.4g tannic acid, add 10mL PBS buffer to prepare a 0.04g / mL tannic acid solution. Add 0.6g uranyl acetate to 20mL water to prepare a 0.03g / mL uranyl acetate solution. Weigh 0.03g potassium permanganate, add 10mL water (solvent) to prepare a 3mg / mL potassium permanganate solution.

[0079] The prepared tannic acid solution, potassium permanganate solution and uranyl acetate were mixed in a volume ratio of 1:3:0.5 to prepare a fixative, wherein the concentration of tannic acid after mixing was 8.89 mg / mL, the concentration of uranyl acetate was 3.33 mg / mL, and the concentration of potassium permanganate was 2.2 mg / mL.

[0080] Example 3

[0081] This example prepares the fixative for ultrathin section electron microscopy

[0082] Prepare virus fixative: Weigh 0.4g tannic acid, add 10mL PBS buffer to prepare a 0.04g / mL tannic acid solution. Add 0.6g uranyl acetate to 20mL water to prepare a 0.03g / mL uranyl acetate solution. Weigh 0.03g potassium permanganate, add 10mL water (solvent) to prepare a 3mg / mL potassium permanganate solution.

[0083] The tannic acid solution, potassium permanganate solution and uranyl acetate prepared above were mixed in a volume ratio of 1:4:1 to prepare a fixative, wherein the concentration of tannic acid after mixing was 6.67 mg / mL, the concentration of uranyl acetate was 5 mg / mL, and the concentration of potassium permanganate was 2 mg / mL.

[0084] Example 4

[0085] This example prepares the fixative for ultrathin section electron microscopy

[0086] Prepare virus fixative: Weigh 0.4g tannic acid, add 10mL PBS buffer to prepare a 0.04g / mL tannic acid solution. Add 0.6g uranyl acetate to 20mL water to prepare a 0.03g / mL uranyl acetate solution. Weigh 0.03g potassium permanganate, add 10mL water (solvent) to prepare a 3mg / mL potassium permanganate solution.

[0087] The tannic acid solution, potassium permanganate solution and uranyl acetate prepared above were mixed in a volume ratio of 1:4:2 to prepare a fixative, wherein the concentration of tannic acid after mixing was 5.71 mg / mL, the concentration of uranyl acetate was 8.57 mg / mL, and the concentration of potassium permanganate was 1.71 mg / mL.

[0088] Application Example 1

[0089] This application example uses ultrathin section electron microscopy to detect cell samples

[0090] Collect SP2 / 0 cell precipitate, add 5% glutaraldehyde to it, fix for 24h, centrifuge slightly, discard supernatant and collect cell mass. Embed the cell mass into a block with agar, wash the cell block with PBS buffer, wash 3 times. Add 0.5mL of fixative prepared in Example 1, fix for 1h, wash the fixed sample with deionized water, wash three times. Dehydrate the sample with gradient ethanol of 30% and 50% for 10min, then treat with 70% ethanol for 6h, dehydrate the sample with gradient ethanol of 90% and 100% for 10min, then add pure acetone for 15min, treat with gradient mixture of acetone and resin with a volume ratio of 2:1 for 1h, then treat with gradient mixture of acetone and resin with a volume ratio of 1:1 and 1:2 for 2h, and finally use pure resin for infiltration for 12h.

[0091] The samples were embedded and polymerized at 45°C for 6 hours. The samples were ultrathinly sliced ​​and stained with 0.03 g / mL uranyl peroxide for 19 minutes and 0.03 g / mL lead citrate for 15 minutes before transmission electron microscopy.

[0092] Application Example 2

[0093] This application example uses ultrathin section electron microscopy to detect cell samples

[0094] Collect SP2 / 0 cell precipitate, add 2% glutaraldehyde, fix for 2h, centrifuge slightly, discard supernatant and collect cell mass. Embed cell mass into block with agar, wash the cell block with PBS buffer, wash 3 times. Add 0.5mL fixative prepared in Example 2, fix for 1h, wash the fixed sample with deionized water, wash three times. Dehydrate the sample with gradient ethanol of 30% and 50% for 10min, then treat with 70% ethanol for 10h, dehydrate the sample with gradient ethanol of 90% and 100% for 10min, then add pure acetone for 20min, treat with gradient mixture of acetone and resin volume ratio of 2:1 for 1h, then treat with gradient mixture of acetone and resin volume ratio of 1:1 and 1:2 for 2h, and finally use pure resin to infiltrate for 12h.

[0095] The samples were embedded and polymerized at 60°C for 12 hours. The samples were ultrathinly sliced ​​and stained with 0.03 g / mL uranyl acetate for 20 minutes and 0.03 g / mL lead citrate for 10 minutes before transmission electron microscopy.

[0096] Application Example 3

[0097] This application example uses ultrathin section electron microscopy to detect virus samples

[0098] The HEK-293 cell culture supernatant sample containing X-Mμlv virus was centrifuged at 80000g for 1h, and the supernatant was discarded to collect the precipitate. The precipitate was made into an agar block and fixed with 5% glutaraldehyde. 1mL of the fixative prepared in Example 3 was added, and the fixed sample was washed with deionized water after fixing for 0.5h, and washed three times. The sample was dehydrated for 10min using gradient ethanol with concentrations of 50%, 70%, 90% and 100%, and then treated with pure acetone for 10min, and treated with gradient mixed solutions with acetone and resin volume ratios of 1:1, 1:2 and 2:1 respectively for 1h, and finally infiltrated with pure resin for 12h.

[0099] The samples were embedded and polymerized at 45°C for 6 hours. The samples were ultrathinly sliced ​​and stained with 0.03 g / mL uranyl acetate for 15 minutes and 0.03 g / mL lead citrate for 15 minutes before transmission electron microscopy.

[0100] Application Example 4

[0101] This application example uses ultrathin section electron microscopy to detect virus samples

[0102] The supernatant sample of HEK-293 cell culture containing X-Mμlv virus was centrifuged at 120000g for 1h, and the supernatant was discarded and the precipitate was collected. The precipitate was made into an agar block and fixed with 2% glutaraldehyde. 0.5mL of the fixative prepared in Example 4 was added, and the fixed sample was washed with deionized water after fixing for 2h, and washed three times. The sample was dehydrated for 10min using gradient ethanol with concentrations of 50%, 70%, 90% and 100%, and then pure acetone was added for 20min, and the gradient mixture with acetone and resin volume ratios of 1:1, 1:2 and 2:1 was used for 1h, and finally pure resin was used for infiltration for 12h.

[0103] The samples were embedded and polymerized at 60°C for 12 hours. The samples were ultrathinly sliced ​​and stained with 0.03 g / mL uranyl acetate for 20 minutes and 0.03 g / mL lead citrate for 10 minutes before transmission electron microscopy.

[0104] Comparative application example 1

[0105] This application example uses an ultrathin section electron microscope method to detect cell samples. The only difference between this application example and application example 1 is that the fixative is replaced with osmic acid, and the rest is the same as application example 1.

[0106] Comparative Application Example 2

[0107] This application example uses an ultrathin section electron microscopy method to detect virus samples. The only difference between this application example and application example 3 is that the fixative is replaced with osmic acid, and the rest is the same as application example 3.

[0108] Test Example 1

[0109] This test case is used to compare the effect of using osmium acid fixative in the application example and ultrathin section electron microscopy

[0110] This test case uses the traditional fixative osmium acid to perform cell and virus fixation detection experiments. First, Figure 1 This is the cell fixation effect diagram of Application Example 1. Figure 2 For comparison of the cell fixation effect diagram of Application Example 1, Figure 3 This is the cell fixation effect diagram of Application Example 2. It can be seen that in the cell ultrathin sectioning method, the cell structure fixed by this reagent scheme has moderate contrast with the cell nucleus and the virus particles in the cell or outside the cell membrane, the virus structure is clearly discernible, and the fixation effect is equivalent to that of osmium acid.

[0111] Secondly, Figure 4 This is a picture of the virus fixation effect of Application Example 3. Figure 5 This is a diagram showing the virus fixation effect of Application Example 4. Figure 6 This is a comparison of the virus fixation effect of Application Example 2; it can be seen that in the ultrathin section electron microscopy technique to detect viruses in cell culture supernatant, by replacing osmium acid as a fixative, the virus particle structure is clear, the contrast is obvious, and it is easy to distinguish from non-viral substances. Moreover, this reagent solution is equivalent to osmium acid fixation, and can successfully identify and detect virus particles in cell culture supernatant.

[0112] In summary, the fixative prepared by the present invention using a combination of tannic acid, potassium permanganate and uranyl acetate has a clear structure and morphology after the sample is fixed in the experiment, which is easy to distinguish, and has a comparable fixation effect with osmium acid. The toxicity of the raw materials is lower than that of osmium acid, and has a wider application scenario.

[0113] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A fixative for ultrathin section electron microscopy, characterized in that: The fixing agent includes tannic acid, potassium permanganate and uranyl acetate.

2. The fixing agent according to claim 1, characterized in that The fixative is divided into a cell fixative and a virus fixative according to the different treatment objects; Preferably, the concentration of tannic acid in the cell fixative is 3.33-13.33 mg / mL; Preferably, the concentration of potassium permanganate in the cell fixative is 1.80-3.64 mg / mL; Preferably, the concentration of uranyl acetate in the cell fixative is 1.82-10 mg / mL.

3. The fixing agent according to claim 2, characterized in that The concentration of tannic acid in the virus fixative is 2.86-13.33 mg / mL; Preferably, the concentration of potassium permanganate in the virus fixative is 1.71-3.64 mg / mL; Preferably, the concentration of uranyl acetate in the virus fixative is 1.82-14.29 mg / mL.

4. A method for preparing a fixative for ultrathin section electron microscopy according to any one of claims 1 to 3, characterized in that: The preparation method comprises the steps of dissolving tannic acid, uranyl acetate and potassium permanganate respectively and then mixing the three solutions.

5. The preparation method according to claim 4, characterized in that: The solvent of the tannic acid comprises PBS buffer; Preferably, the concentration of the tannic acid after dissolution is 0.02-0.06 g / mL; Preferably, the solvent of the uranyl acetate comprises water; Preferably, the concentration of the uranyl acetate after dissolution is 0.02-0.05 g / mL; Preferably, the solvent of potassium permanganate includes water; Preferably, the concentration of the potassium permanganate after dissolution is 3-5 mg / mL; Preferably, the volume ratio of tannic acid, potassium permanganate and uranyl acetate in the cell fixative is 1:(3-4):(0.5-1.0); Preferably, the volume ratio of tannic acid, potassium permanganate and uranyl acetate in the virus fixative is 1:(3-4):(0.5-2.0).

6. A detection method using an ultrathin section electron microscope, characterized in that: The detection method comprises sample pretreatment, fixing the sample with a fixative used in ultrathin section electron microscopy according to any one of claims 1 to 3, washing, dehydration, penetration, embedding and polymerization, and then ultrathin sectioning, and staining the section before detection.

7. The detection method according to claim 6, characterized in that: The sample comprises a cell sample and / or a virus sample; Preferably, the cell sample pretreatment step comprises fixing the cell sample with glutaraldehyde, collecting cell clusters, embedding them into blocks with agar, and then washing them; Preferably, the concentration of glutaraldehyde in the pretreatment of the cell sample is 2-5%; Preferably, the washing solution comprises PBS buffer; Preferably, the virus sample pretreatment step comprises collecting the precipitate after centrifuging the sample, making the precipitate into an agar block and fixing it with glutaraldehyde; Preferably, the centrifugation time is 1-2h, and the centrifugation speed is 80000-120000g; Preferably, the concentration of glutaraldehyde in the virus sample pretreatment is 2-5%.

8. The detection method according to claim 6 or 7, characterized in that: The volume of the fixative added during the fixation is 0.3-1.0 mL; Preferably, the fixing time is 0.5-2h; Preferably, the cleaning reagent includes deionized water; Preferably, the dehydration and infiltration step comprises: dehydration treatment with gradient ethanol, followed by acetone treatment, followed by treatment with gradient mixed solution, and finally infiltration with pure resin; Preferably, the concentration range of the gradient ethanol of the virus sample is 50-100%; Preferably, in the gradient ethanol dehydration treatment of the virus sample, the dehydration time of each gradient ethanol is 10-20 min; Preferably, the concentration range of the gradient ethanol of the cell sample is 30-50%; Preferably, in the gradient ethanol dehydration treatment of the cell sample, the dehydration time of each gradient ethanol is 10-20 min; Preferably, the cell sample is subjected to a second dehydration treatment and a third dehydration treatment after the dehydration treatment; Preferably, the reagent for the second dehydration treatment is 60-80% ethanol; Preferably, the second dehydration treatment time is 6-12h; Preferably, the reagent for the third dehydration treatment is 90-100% gradient ethanol; Preferably, in the third dehydration treatment, the dehydration time of each gradient ethanol is 10-20 min; Preferably, the acetone treatment time is 10-20 min; Preferably, the gradient mixed solution comprises acetone and resin; Preferably, the volume ratio of acetone to resin in the gradient mixed solution is (1-2):(1-2); Preferably, the re-treatment time is 1-2h; Preferably, the penetration time is 6-12 hours.

9. The detection method according to any one of claims 6 to 8, characterized in that: The embedding polymerization temperature is 45-60°C and the time is 6-12h; Preferably, the dyeing step comprises dyeing with uranyl acetate and then dyeing with lead citrate; Preferably, the concentration of uranyl acetate is 0.02-0.05 g / mL; Preferably, the uranyl acetate staining time is 15-20 min; Preferably, the concentration of the lead citrate is 0.02-0.05 g / mL; Preferably, the staining time of the lead citrate is 10-15 min.

10. Use of a fixative for ultrathin section electron microscopy according to any one of claims 1 to 3 or a detection method using an ultrathin section electron microscope according to any one of claims 6 to 9 in ultrathin section electron microscopy detection.

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