Electron microscope observation experiments on cultured cells
Electron microscope observation experiments on cultured cells
Cultured cells are characterized by thin layers and fresh material, which are easily penetrated during fixation, and are well fixed and suitable for electron microscopic observation. Content source: tissue culture and molecular cytology techniques. (Beijing Publishing House)
Operation method
Electron microscope observation experiments on cultured cells
Principle
To do transmission electron microscopy observation need to be sliced, fixed and embedded cells process, and general tissue sectioning method is largely similar to the biggest problem is how to cells intact from the wall of the culture flask embedded down. This difficulty has been overcome since the introduction of the stereotyped product, plastic film. The application of sterile plastic film is an extremely desirable support, better than the use of coverslips, microporous filter paper, mica and eggshell membrane and other supports. Cultivate the cells directly on the plastic film, not only the cell growth effect is good and can be embedded with the cells together with the section, eliminating a lot of trouble. If the use of other materials is not only unfavorable to cell growth, the biggest drawback is that the cells need to be separated from the support before embedding, increasing the workload.
Materials and Instruments
Cells Move I. Transmission observation method For more product details, please visit Aladdin Scientific website.
Glutaraldehyde PBS Isoamyl acetate
Glass vials Centrifuge Microscope
Transmission observation must be sliced, according to the type of cultured cells and culture methods are divided into two kinds of in situ sections and digested sections. According to the different types of cultured cells and culture methods, there are two kinds of slices: in situ slice and digested slice.
1. It is very complicated to make in-situ section when cells are cultured in glass vials for transmission observation. It is very complicated to do in situ sectioning when cells are cultured in glass vials for transmission observation. The process is as follows: digestion and isolation of cells, digestion and isolation of cells. The process is as follows: digestion and separation of cells, fixation, embedding, sectioning and observation. The process is as follows: digestion and isolation of cells, fixation, embedding, sectioning and observation.
When digestion is carried out, attention should be paid to the concentration of the digestive solution should not be too large, the digestion time should be moderate, and the blowing action of the cells should be slight to prevent damage to the microstructure of the cell surface. The cells should be blown gently to prevent damage to the microstructure of the cell surface. The cells should then be centrifuged at a low speed to the bottom of the centrifuge tube (a cone-shaped centrifuge tube is best). Then centrifuge the cells at a low speed, so that the cells sink to the bottom of the centrifuge tube (a conical centrifuge tube is the best), aspirate the supernatant, and immediately add glutaraldehyde fixation. The rest of the embedding and sectioning steps are the same as those for general tissues. The rest of the embedding and sectioning steps are the same as those for general tissues, please refer to other electron microscopy techniques, which will not be repeated here.
2. The advantage of the cell embedding method is that a large number of cells can be obtained, which makes embedding and sectioning easier.
The disadvantage is that digestion may damage the surface structure of the cells, and the second is that digestion alters the relationship of the cells to each other when the cells are grown in a monolayer. The disadvantage is that digestion may damage the surface structure of the cells, and the second is that digestion changes the relationship between the cells during monolayer growth.
3. In situ embedding can also be used, for which the cells should be cultured on sterile polystyrene plastic film, which is convenient for fixation, embedding and sectioning. for fixation, embedding, and sectioning.
Polystyrene film is a thick cover sheet made of polystyrene, Non-toxic, soft texture, sterilized packaging of finished products (domestic production has not yet seen). It can be cut into suitable small pieces and placed into the bottle, and then inoculate the cells into the bottle, after the cells attach to the cap sheet and grow, remove, fix, cut into After the cells are attached to the capsule and grow, they can be removed, fixed, cut into small pieces, and directly embedded into Epon capsules, which are easy to be sliced, and it is an ideal method for in situ embedding and observation of cells by transmission electron microscopy.
It is an ideal method for in situ embedding and transmission electron microscopy observation of cells. If the observation is of suspension cells, because the suspension cells do not need to be digested, they can be centrifuged, fixed, embedded and sliced directly. and slicing, the cell preservation effect is good.
Scanning observation
Scanning observation of cultured cells is very convenient, in the observation of wall cells also need to be digested, the preparation of After preparing the cell suspension, rinse it with Hanks' solution for 1 or 2 times to remove cell fragments and serum proteins (so as not to hinder the observation). The rest of the treatment is the same as that of the blood cell scanning electron microscopy observation method.
In situ scanning of cultured cells is easier and can be performed as follows:
1. add support small capsule culture method to culture the cells to the exponential proliferation stage;
2. PBS pH 7.4 rinsed twice to remove culture fluid;
3. 25 % glutaraldehyde/PBS fixation for 30 minutes. 4;
4. PBS rinsed 3 times;
5. 1 % OsO4 fixation for 45 minutes. 6;
6. rinsing with PBS 3 times;
7. dehydration
Acetone/isoamyl acetate (1:1) for 10 minutes → isoamyl acetate for 30 minutes. 8. critical point drying;
8. critical point drying;
9. gold spraying;
10. scanning electron microscopy (Hitachs-450), imaging.