This experiment is mainly used for fixing frozen sections.

Operation method

Frozen section fixation experiment

Principle

Frozen sections used in in situ hybridization must be fixed with paraformaldehyde and then dehydrated.

Materials and Instruments

Frozen Tissue
PFA PBS Ethanol
Humidifying cartridge

Move

1. Place the slide containing the section into a humidifying box, add 4% PFA to cover the entire section, cover the box, and leave at room temperature for 20 min for pre-fixed tissues, or 5 min for fixed tissues.

2. Aspirate off the fixative, rinse the sections with 3xPBS using a capillary pipette or a squirt bottle and incubate for 5 min. 3.

3. Repeat step 2 with 1xPBS, aspirate the PBS, and rinse the sections with 30%, 60%, 80%, 95%, and 100% ethanol sequentially for 2 min each time.

4. Dry the slides and store them in a sealed box with desiccant at -70°C for up to several weeks. Equilibrate to room temperature before opening the box.

Figure 1. Humidifying box

Caveat

1. Timely fixation is the first element in the production of frozen sections, and the best time for fixation should be immediately after sectioning, when the slides of the mounted tissue sections are put into the fixative solution for fixation. The fixative should be kept fresh and changed frequently.

2. Methanol fixation time is fast, cell shrinkage is small, and the nucleus and antigen are better preserved. 95% ethanol is a fixative with dehydrating effect, weak penetration, slow fixation, good for glycogen protein fixation, can dissolve lipids, easy to cause tissue cell shrinkage. Acetone can make protein precipitation, strong penetration, serious cell contraction, relatively strong fixation of cell nucleus, better fixation of enzymes, the role is equivalent to ethanol, there is no fixation of glycogen.AF liquid is a mixture of ethanol and formaldehyde liquid fixative, both ethanol and formaldehyde liquid, fixation and at the same time have the effect of dehydration.AAF liquid and AF liquid fixation liquid role is similar to the fixation of the role of dehydration at the same time. Because of more glacial acetic acid, it can fix the chromatin quickly, and the fixation effect is uniform, but also can offset part of the contraction effect of ethanol on the tissue.

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