Immunoelectron microscopy specimens need to be fixed after sampling. Source of content Immunohistochemistry Laboratory Techniques and Applications

Operation method

basic program

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1. Vascular perfusion fixation has the best fixation effect and enables good preservation of ultrastructure. The perfusion device consists of a pressurized balloon, a pressure gauge, a three-way connector and connecting tubing. Different tissues and organs must choose the appropriate perfusion pressure, see the relevant books for details. Perfusion fixation is usually maintained for 10-15 min, and then continue immersion fixation with the same fixative solution for 2-4 h. 2.

2. immersion fixation of tissue pieces cut into small pieces and immediately put into the fixation solution, placed in 4 ℃ fixed for 2 ~ 4 h. Free culture cells centrifuged to remove the supernatant, add 20 times the volume of the fixation solution, use a pipette to break up the cell clusters, fixed for 25 ~ 30 min. 3. microwave fixation can accelerate the speed of the cell clusters.

3. Microwave fixation Microwave can accelerate the movement and diffusion of internal molecules and external chemical substances in tissues. The application of microwave irradiation can shorten the fixation time, and the antigen can be better preserved. Generally 700 W microwave irradiation for 10 s, put 1000 ml of cold water in the microwave oven to absorb the heat (keep the temperature below 35 ℃), and then continue to fix with the fixative for 2 to 4 hours.

Common Problems

At present, the following fixatives are commonly used for immunoelectron microscopy specimens.

1. paraformaldehyde-glutaraldehyde mixed fixative (referred to as PG fixative) is recommended to use 2% to 4% paraformaldehyde and 0.5% glutaraldehyde mixture, the fixation time is 4 hours.

2. periodate - lysine - paraformaldehyde mixed fixative (PLP fixative) preparation method, see chapter 1 of this book, fixation time of 6 ~ 12 h. The fixative selectively fixation of sugar, the ultrastructure and many antigens of the activity of the preservation of good. The low concentration of paraformaldehyde can stabilize proteins and lipids.

3. Picric acid-polyformaldehyde-glutaraldehyde fixative (PAPG fixative) contains 4% paraformaldehyde, 15% (v/v) picric acid (2,4,6-trinitrophenol), 0.5% glutaraldehyde, pH 7.3. Picric acid has a strong penetrating power and has little effect on the antigenic activity. Adding picric acid to the pre-fixation solution instead of osmiic acid can immobilize proteins and improve the preservation of membranes and cytoplasm.

Commonly used in immunostaining after embedding.

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