Very straightforward.

Make sure brain sections have been thoroughly dried, at least overnight at 40°C. If struggling with sections floating off give them a bake for half an hour at 60°C before moving to next step.

3 x 5 mins in xylene- go longer if you're still seeing waxy deposits, it won't do any harm.

3 x 5 mins in IMS

5 mins 90% IMS

5 mins 80% IMS

5 mins 70% IMS

water

We tend to use citrate buffer (pH 6) as a starting point for AR. Preheat buffer in closed container in a steamer (or whatever heating method is available to you as long as the buffer isn't allowed to boil) then slides in and heated for 25 mins then allowed to cool down slowly.

This is a very successful method for both IHC and IF in our lab. Happy to answer further questions.

Thank you! James Matthew Minshull

@james Matthew Minshull

Please I am currently working on FFPE mouse brain for IF, I have before having difficulty on antibodies signal. The signal is only observed at the surface of the cell and not anywhere else. We are suspecting a case of antibodies not penetrating. The tissue was fixed in NBF for 48hrs, could it be a case of over fixation? If yes, do you think it can be reversed? Any protocol for removing excess fixative in brain section will be appreciated too