Hey Sangeeta,

my experiences with this type of sample is also not very good. I did immunofluorescence and the signal is really weak. However, I will share what little improvement suggestions I can offer: The slides were cut and then immediately stored at -80°C until staining. Post-fixation of the sections for 10 min worked ok with methanol or Aceton/Ethanol (1:1). I compared 4 °C with -20 °C and saw only little difference. So in the end, I decided for Aceton/Ethanol (less toxic and a bit better) at -20 °C (I store it in the freezer). Afterwards leave the sections at room temperature until dry, encircle and move on with your staining.

Post fixation with PFA or Aceton alone destroyed the morphology of certain markers. I also tried antigen retrieval (pH6 and pH9) and (despite common suggestions) it enhanced the signal for some markers, but seems to weaken others, so it depends on what you want to stain.

Antibody staining with overnight incubation at 4 °C worked better than 1h at room temperature. But just as warning: in the end, all the images still didn't look pretty.

If you don't mind me asking: What do you want to stain?

Best,

Theresa

If mouse or rat, you can perfuse the animal with 4%PFA that is common procedure.

I perfused mouse with 4% PFA, collected brain, leave brain Overnight in 4%PFA at 4C. Than you can freeze. After frozen sections are cut. Warm section to 4C, treat with 0.2% glutaraldehyde for 10 min, wash with PBS 3/4 times with PBS and go ahead with your IHC. That was my protocol. I followed. ISH, IHC, IF procedures works on these sections.

Thank you very much Both of you actually, I have worked with various primary antibodies for IHC,IF for perfused animal but now we want to stain with various primary antibodies with the snap frozen brain sections already cut(12microns) for MSI I know it's hard to get done for IHC but looking for right fixation method before I can do IHC.

Good luck