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IHC frozen sections staining protocol

互联网2013-11-13

1013

实验步骤

Frozen sections: Once mounted on APES coated slides, frozen sections are best kept at -80°C until needed.

1. When required, leave to warm at room temperature for 5 min.

2. Pre cool the fixative (acetone, methanol or ethanol) at -20°C for 30 min. (Abcam recommends starting with acetone)

3. Fix with the pre cooled fixative for 5-10 min, at room temperature.

4. Rinse 3-4 X in PBS.

5. Continue with the immunohistochemical staining protocol.

If the tissue samples are fixed with an aldehyde fixative such as formalin, paraformaldehyde or glutaraldehyde and immunofluorescence (IF) is the detection method, consider including 0.3 M glycine in the blocking buffer, before applying the primary antibody. Glycine will bind free aldehyde groups that would otherwise bind the primary and secondary antibodies, leading to high background. Background due to free aldehyde groups is more likely to occur when the fixative is glutaraldehyde or paraformaldehyde.

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