This depends on your aims of staining. If your antibody is sensitive to formalin-fixation, frozen sections would be recommended. If you do an IHC-staining with a FFPET-antibody, it would be recommended to fix in NBF or PFA for a definite time (12-48 hours), then process to paraffin and store the blocks until staining.

For immunofluorescence frozen sections are mostly preferred because of unspecific background staining of FFPET tissue.

This depends on your aims of staining. If your antibody is sensitive to formalin-fixation, frozen sections would be recommended. If you do an IHC-staining with a FFPET-antibody, it would be recommended to fix in NBF or PFA for a definite time (12-48 hours), then process to paraffin and store the blocks until staining.

For immunofluorescence frozen sections are mostly preferred because of unspecific background staining of FFPET tissue.

Thank you Gudrun and Mohamed

Sravan,

I agree with Gudrun, if you go for PFA you should do this for a definite time.

Another point is that the morphology in frozen sections will be comparably bad.

Some antibodies don't work that well after paraffine treatment. Therefore it is very common to always freeze the tissue, but not flash freezing, that destroys the morphology. This is how we do:

1. perfuse the animal with PBS followed with PFA

2. dissect out your tissue of interest and post-fix it in PFA for a defined time, usually 2-12 hours.

3. Put it in a solution of 10% sucrose overnight or until it sinks to the bottom

4. Put it in a solution of 20% sucrose overnight or until it sinks to the bottom

5. Put it in a solution of 30% sucrose overnight or until it sinks to the bottom

6. Embed it in OCT and freeze, for example on dry ice. Store in the freezer, section on cryostat.

Thank you Marina. I appreciate the details. It is really helpful.

I agree with Marina, that's the method we have used for freezing mouse tumours for histology. Is it just paw tissues or are there still bones in th etissue (ie whole paw?) in which case you will need to decalcify the paws before trying to section them! We usually use the EDTA method for that but there are several to try.