For H&E staining and immunohistochemistry studies of brain tissue, we performed trans-cardiac perfusion with 4 % formaldehyde and then post-fixing in 4% formaldehdye for 24 hours. But I could not get good sectioning, tissue separated from paraffin. Is it related with perfusion time or anything else?
Just remember penetrartion time of 4% formaldehyde. It is 1mm/1 hour. So co-relate kindly it with the size of rat brain. However, I think 4% formaldehyde is not suitable to fix brain and its efficiency is not appropriate in brain biopsies.
So look to some brain suitable fixative and fix brain in that.
I agree. It sounds like your porblem is with the embedding and not the fixation. You may want to increase the time of paraffin infiltration.
keep brain in 4% PFA for 2-3 hours only after perfusion then transfere in 30% sucrose in fredge till time of using
Before cutting the praffin block, keep it on cold surface for at least 30 min will help cutting
Thank you for all. I fixed the problem with increasing the time of paraffin infiltration. Although I got good sections (6 microns), sections got away from the slides in water step of H&E staining. What could be the reason of that? Could it be related with the increased time for paraffin infiltration or else?
Which slide adhesive you are using?
It is slide adhesive which fixes section to slide.
We do not use paraffin embedding, but have found letting the tissue dry (but not dry out) after antigen retrieval and PBS washes keeps the tissue adhered to the slides better throughout. We usually do this in a stain tray by adding water to the reservoir but leaving the lid off. (We use: Stain Tray Slide staining system with black lid (Electron Microscopy Sciences 71397-B $201 http://www.emsdiasum.com/microscopy/products/histology/staining.aspx))
Good luck!
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