Oocyte transmission electron microscope sample preparation method

Abstract

The invention discloses an oocyte transmission electron microscope sample preparation method, and belongs to the technical field of electron microscope sample preparation. On the basis of conventional ultrathin section technology sample preparation, a single or a very small amount of oocytes are dyed with eosin and then are pre-coated with a human granular cell line, so that the form of the oocytes is maintained, and preparation of a high-quality oocyte transmission electron microscope sample is realized; the visualization of the oocyte and the operability of the whole sample are realized in the sample slicing process, so that blind cutting and cell loss are avoided, the preparation process of the oocyte sample is optimized, the success rate of the transmission electron microscope sample of the oocyte is improved, and the detection efficiency is improved. A solution is provided for high-quality electron microscope sample preparation of very few precious oocytes and other rare cells of human or mice, so that ultrastructure observation and pathological diagnosis become possible.

Description

Technical Field

[0001] The invention relates to the technical field of electron microscope sample preparation, in particular to an oocyte transmission electron microscope sample preparation method. Background Art

[0002] Oocyte quality is a key factor in determining pregnancy outcomes, and the correct conduct of meiosis is the basic condition for ensuring oocyte quality. Various organelles are the guarantee for the correct conduct of meiosis, so the ultrastructure of various organelles is crucial to oocyte quality and pregnancy outcomes.

[0003] Transmission electron microscopy is one of the important means of observing ultrastructure, but oocytes are few in number and difficult to obtain, and the preparation process of transmission electron microscopy samples is complicated, and cells are easily lost or damaged, which leads to high production costs of oocyte transmission electron microscopy samples. Conventional oocyte transmission electron microscopy samples contain only one or a few cells, and the process of finding target cells is difficult, which causes trouble for the observation of oocyte ultrastructure. Therefore, providing a transmission electron microscopy sample preparation method with a high sample preparation success rate, high cell structure integrity, and easy observation is of great significance to the study of the ultrastructure of oocytes and rare cells. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing oocyte transmission electron microscopy samples to solve the problems existing in the above-mentioned prior art. Based on the conventional ultrathin sectioning technology, the present invention uses a human granulosa cell line to pre-coat a single or very small amount of oocytes after staining with eosin to maintain the morphology of the oocytes, thereby realizing the preparation of high-quality oocyte transmission electron microscopy samples, and realizing the visualization of oocytes and the operability of the overall sample during the sample sectioning process, avoiding blind cutting and cell loss, optimizing the oocyte sample preparation process, and improving the success rate of oocyte transmission electron microscopy samples. It provides a solution for the preparation of high-quality electron microscopy samples of very small amounts of precious oocytes and other rare cells of humans or mice, making their ultrastructure observation and pathological diagnosis possible.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an oocyte transmission electron microscope sample preparation method, comprising the following steps:

[0007] (1) Fix the oocytes in glutaraldehyde fixative;

[0008] (2) transferring the fixed oocytes into eosin staining solution for staining;

[0009] (3) transferring the stained oocytes into a human ovarian granulosa cell resuspension solution, centrifuging and collecting the precipitate;

[0010] (4) adding the precipitate to a glutaraldehyde fixative for fixation and rinsing;

[0011] (5) adding the rinsed precipitate into an osmium acid solution for fixation and rinsing;

[0012] (6) Dehydration and osmosis;

[0013] (7) Ultrathin sections;

[0014] (8) Double staining the ultrathin sections.

[0015] Optionally, in step (1), the concentration of the glutaraldehyde fixative is 2.5%-3%; the fixation temperature is 2-4°C, and the fixation time is 25-35 minutes.

[0016] Furthermore, in step (2), the dyeing temperature is 20-25° C. and the dyeing time is 8-12 min.

[0017] In the present invention, by performing eosin staining on oocytes, visualization of oocytes in subsequent sample preparation is achieved, loss of oocytes in processes such as centrifugation, fixation, rinsing, dehydration, and penetration is avoided, and the utilization rate of oocytes in the transmission electron microscope sample preparation process is improved.

[0018] Optionally, in step (3), the ratio of the number of human ovarian granulosa cells to the number of stained oocytes is 10 6-8 : 1; the centrifugal speed is 800-1500rpm, and the time is 4-6min.

[0019] In the present invention, the human ovarian granulosa cell resuspension is a digested human ovarian granulosa cell resuspension. Compared with the traditional method of pre-embedding with materials such as agar or egg white, by transferring the oocytes into the human ovarian granulosa cell resuspension and embedding the oocytes with cells in a natural state, the damage to the morphology of the oocytes during the subsequent sample preparation process, such as centrifugation, fixation, and rinsing, can be reduced.

[0020] Optionally, in step (4), the concentration of the glutaraldehyde fixative is 2.5%-3%; the fixation temperature is 2-4°C, and the fixation time is 12h-16h.

[0021] Optionally, in step (5), the concentration of the osmium acid solution is 1%-2%; and the fixation time is 1-2h.

[0022] Optionally, in step (6), the dehydration method is: dehydrating step by step with ethanol solutions of volume fractions of 30%, 50%, 70%, 90%, 100%, and 100%, each step for 20 minutes, and finally replacing with propylene oxide.

[0023] Optionally, in step (6), the infiltration method is: infiltrating in a mixture of Epon812 resin and acetone in a volume ratio of 1:1 for 2-4 hours, and then infiltrating in Epon812 embedding solution for 12-16 hours; then placing the cell precipitate sample at one end of the embedding plate well and polymerizing;

[0024] The polymerization method is: polymerization at 37° C. for 12-24 h, and then polymerization at 60° C. for 12-24 h.

[0025] Optionally, in step (7), the thickness of the ultrathin section is 50-100 nm.

[0026] Optionally, in step (8), the double staining method is: double staining the ultrathin sections using uranyl acetate staining solution and lead citrate staining solution with a mass percentage concentration of 1%-3%.

[0027] The present invention discloses the following technical effects:

[0028] The present invention improves the electron microscope sample preparation method of a single or very few oocytes, realizes the visualization of oocytes in the sample preparation and slicing process by performing eosin staining on oocytes, and avoids blind cutting and sample loss to the greatest extent; the oocytes are pre-coated with KGN cells, and the method of pre-coating with cells in a relatively natural state can reduce the morphological damage of oocytes caused by centrifugation and the like in the sample preparation process, so that the fixed oocytes can maintain their original morphology to the greatest extent, improve the sample quality, and realize the operability of extremely trace samples.

[0029] The method for preparing oocyte electron microscopic samples of the present invention improves the defects of damaged cell structure or unclear observation field caused by traditional agar or egg white, and provides a solution for high-quality preparation and efficient observation of single or very few oocyte electron microscopic samples. It can be used for the preparation of high-quality electron microscopic samples of very small amounts of precious oocytes and other rare cells of humans or mice, making their ultrastructural observation and pathological diagnosis possible. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 This is an electron micrograph of the transmission electron microscopy sample of the oocyte prepared in Example 1, with a scale bar of 10 μm;

[0032] Figure 2This is an electron micrograph of the transmission electron microscopy sample of the oocyte prepared in Example 2, with a scale bar of 10 μm. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] Example 1

[0039] This embodiment takes mouse oocytes as an example to provide a method for preparing oocyte transmission electron microscopy samples, and the specific process is as follows:

[0040] 1. Under a stereomicroscope, transfer a single oocyte to 1 mL of 3% glutaraldehyde fixative and fix at 4°C for 30 min.

[0041] 2. Under the stereomicroscope, gently rinse the oocytes with PBS to wash off the fixative, and transfer the oocytes to eosin staining solution and stain at room temperature for 10 minutes.

[0042] 3. Under the stereomicroscope, gently rinse the oocytes with PBS to wash away the dye.

[0043] 4. Digest the KGN cells until the cells are dispersed and resuspend them. Transfer the oocytes to the resuspension solution of the KGN cell line (containing 1×10 KGN cells). 7 The supernatant was discarded and the precipitate was gently washed with PBS, and the mixture was centrifuged again (1000 rpm, 5 min), the supernatant was discarded and the precipitate was collected.

[0044] 5. Add 2 mL of 3% glutaraldehyde fixative to the precipitate and fix it overnight at 4°C.

[0045] 6. After fixation, gently pick up the mixed precipitate and rinse the precipitate gently with PBS 3 times, 10 minutes each time.

[0046] 7. Fix the precipitate with 1% osmium acid fixative for 60 minutes, and gently rinse the precipitate with PBS three times, 10 minutes each time.

[0047] 8. Dehydrate step by step with ethanol solutions of volume fractions of 30%, 50%, 70%, 90%, 100%, and 100%, each step for 20 minutes, and finally replace with propylene oxide.

[0048] 9. After dehydration, infiltrate in a 1:1 mixture of Epon812 resin and acetone for 4 hours, and then infiltrate in Epon812 embedding solution overnight (16 hours); then embed the sample, place it in the embedding plate well, place the cell pellet sample at one end of the embedding plate well, and polymerize: polymerize at 37°C for 24 hours, then polymerize at 60°C for 24 hours.

[0049] 10. Trim and slice into ultra-thin sections with a thickness of 50 nm.

[0050] 11. Double stain the ultrathin sections with 1-3% uranyl acetate staining solution and lead citrate staining solution. After natural drying, observe and photograph under a transmission electron microscope. See the electron microscope image for details. Figure 1 .

[0051] Example 2

[0052] This embodiment takes mouse oocytes as an example to provide a method for preparing oocyte transmission electron microscopy samples, and the specific process is as follows:

[0053] 1. Under a stereomicroscope, transfer a single oocyte to 1 mL of 3% glutaraldehyde fixative and fix at 4°C for 30 min.

[0054] 2. Under the stereomicroscope, gently rinse the oocytes with PBS to wash off the fixative, and transfer the oocytes to eosin staining solution and stain at room temperature for 10 minutes.

[0055] 3. Under the stereomicroscope, gently rinse the oocytes with PBS to wash away the dye.

[0056] 4. Digest the KGN cells until the cells are dispersed and resuspend them. Transfer the oocytes to the resuspension solution of the KGN cell line (containing 1×10 KGN cells). 7 The supernatant was discarded and the precipitate was gently washed with PBS, and the mixture was centrifuged again (1000 rpm, 5 min), the supernatant was discarded and the precipitate was collected.

[0057] 5. Add 2 mL of 3% glutaraldehyde fixative to the precipitate and fix it overnight at 4°C.

[0058] 6. After fixation, gently pick up the mixed precipitate and rinse the precipitate gently with PBS 3 times, 10 minutes each time.

[0059] 7. Fix the precipitate with 1% osmium acid fixative for 60 minutes, and gently rinse the precipitate with PBS three times, 10 minutes each time.

[0060] 8. Dehydrate step by step with ethanol solutions of volume fractions of 30%, 50%, 70%, 90%, 100%, and 100%, each step for 20 minutes, and finally replace with propylene oxide.

[0061] 9. After dehydration, infiltrate in a 1:1 mixture of Epon812 resin and acetone for 4 hours, and then infiltrate in Epon812 embedding solution overnight (12 hours); then embed the sample, place it in the embedding plate well, place the cell pellet sample at one end of the embedding plate well, and polymerize: polymerize at 37°C for 24 hours, then polymerize at 60°C for 24 hours.

[0062] 10. Trim and slice into ultra-thin sections with a thickness of 100 nm.

[0063] 11. Double stain the ultrathin sections with 1-3% mass percent uranyl acetate staining solution and lead citrate staining solution. After natural drying, observe and photograph under a transmission electron microscope. See the electron microscope image for details. Figure 2 .

[0064] from Figure 1 and Figure 2 It can be seen that under the electron microscope, the morphology of oocytes and KGN cells is obviously different: the volume of oocytes is significantly larger than that of KGN cells; KGN cells are closely connected, but there is a clear "boundary" between them and oocytes; the field of view of oocytes is clear, the boundaries are complete, the zona pellucida is obvious, the structure of oocytes is complete, and the morphology is clearly visible. This shows that using KGN cells to pre-embed oocytes and prepare transmission electron microscope samples can intuitively and quickly find the location of oocytes, and the obtained oocytes have complete structures, clear fields of view, and are easy to observe.

[0065] The oocyte transmission electron microscopy sample preparation method of the present invention has the advantages of simple operation, easy location of oocytes, and complete oocyte morphology, clear structure, and obvious "boundaries". In the traditional sample preparation method using agar pre-embedding, the solidification speed and degree of agar may be affected by factors such as temperature and pH value, and the pressure generated during the solidification process may cause the cell morphology to change or rupture; in the traditional sample preparation method using egg white pre-embedding, egg white has electron density, which may cause background interference during electron microscopy observation, causing blurred vision. In order to reduce background interference, the sample needs to be fully stained and cleaned, which increases the complexity and time cost of the preparation process. Therefore, the oocyte transmission electron microscopy sample preparation method of the present invention provides a new empirical method for the preparation of high-quality electron microscopy samples of oocytes and the preparation of electron microscopy samples of rare cells.

[0066] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing oocyte samples for transmission electron microscopy, characterized in that: The steps include: (1) Fix the oocytes in glutaraldehyde fixative; (2) transferring the fixed oocytes into eosin staining solution for staining; (3) transferring the stained oocytes into a human ovarian granulosa cell resuspension solution, centrifuging and collecting the precipitate; (4) adding the precipitate to a glutaraldehyde fixative for fixation and rinsing; (5) adding the rinsed precipitate into an osmium acid solution for fixation and rinsing; (6) Dehydration and osmosis; (7) Ultrathin sections; (8) Double staining the ultrathin sections.

2. The oocyte transmission electron microscopy sample preparation method according to claim 1, characterized in that: In step (1), the concentration of the glutaraldehyde fixative is 2.5%-3%; the fixation temperature is 2-4°C, and the fixation time is 25-35 minutes.

3. The method for preparing oocyte samples for transmission electron microscopy according to claim 1, characterized in that: In step (2), the dyeing temperature is 20-25°C and the dyeing time is 8-12 minutes.

4. The method for preparing oocyte samples for transmission electron microscopy according to claim 1, characterized in that: In step (3), the ratio of the number of human ovarian granulosa cells to the number of stained oocytes is 10 6-8 : 1; the centrifugal speed is 800-1500rpm, and the time is 4-6min.

5. The method for preparing oocyte samples for transmission electron microscopy according to claim 1, characterized in that: In step (4), the concentration of the glutaraldehyde fixative is 2.5%-3%; the fixation temperature is 2-4°C, and the fixation time is 12h-16h.

6. The method for preparing oocyte samples for transmission electron microscopy according to claim 1, characterized in that: In step (5), the concentration of the osmium acid solution is 1%-2%; and the fixation time is 1-2h.

7. The method for preparing oocyte samples for transmission electron microscopy according to claim 1, characterized in that: In step (6), the dehydration method is: dehydrating step by step with ethanol solutions of volume fractions of 30%, 50%, 70%, 90%, 100%, and 100%, each step for 20 minutes, and finally replacing with propylene oxide.

8. The method for preparing oocyte samples for transmission electron microscopy according to claim 1, characterized in that: In step (6), the infiltration method is: infiltrating in a mixture of Epon812 resin and acetone in a volume ratio of 1:1 for 2-4 hours, and then infiltrating in Epon812 embedding solution for 12-16 hours; then placing the cell pellet sample at one end of the embedding plate well and polymerizing; The polymerization method is: polymerization at 37° C. for 12-24 h, and then polymerization at 60° C. for 12-24 h.

9. The method for preparing oocyte samples for transmission electron microscopy according to claim 1, characterized in that: In step (7), the thickness of the ultrathin section is 50-100 nm.

10. The method for preparing oocyte samples for transmission electron microscopy according to claim 1, characterized in that: In step (8), the double staining method is: using uranyl acetate staining solution and lead citrate staining solution with a mass percentage concentration of 1%-3% to double stain the ultrathin sections.

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