Dear Monokesh,

I would just drop them straight into 4% PFA. I do brain slices and I only fix them for 15-20 minutes in 4%PFA at RT. Basically I cut vibratome sections and do elecrtrophysiology on them for many hours and then fix them, and it works everytime. Make sure your 4%PFA is fresh or frozen vials. 1 ml vial per slice is plentiful in little petri-dishes. For technical details see my papaers listed below.

best wishes, Refik

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Dear Monokesh,

I would just drop them straight into 4% PFA. I do brain slices and I only fix them for 15-20 minutes in 4%PFA at RT. Basically I cut vibratome sections and do elecrtrophysiology on them for many hours and then fix them, and it works everytime. Make sure your 4%PFA is fresh or frozen vials. 1 ml vial per slice is plentiful in little petri-dishes. For technical details see my papaers listed below.

best wishes, Refik

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Article Neurobiotin Electroporation for Combined Structural and Func...

 Thanks Refik, I was also thinking like this but from literature I found some confusing thoughts. Some has said 4%PFA in PBS for one hour and on the other hand some 4% PFA, PBS also other solution as well for 2 hours thats why I posed that question. Now, I should be clear I think. Thanks for your help. Be fine and will ask more  in the future thats for sure. Bye

Hey Monokesh,

in my studies I did the fixation of brain slices with ice cold 4%PFA on ice for 15 minutes. You may check which option gives the best result for your staining.

best wishes,

Christina

 Thanks a lot Christina. 

we use 4 % PFA in PBS   for 2 hours  at room temperature on a shaking device - best wishes Monika