I'm not familiar with the use of sodium sulfide during perfusion, but I'll assume it is due to one of the targets you intend to look at. I do have extensive experience in 4% PFA perfusions and cryosectioning of mouse brains to look at hippocampal sections. To me, it seems that the tissue integrity is compromised and this usually arises from issues with the fixation. I have also seen this when the brain was freeze-thawed too many times. Since it doesn't look like you freeze-thawed anything, it is likely a fixation issue. Could you provide more details about how you perform the perfusion itself?

The tissue does not look so bad. What kind of staining you have used? If you just need an overview of your brain I would recommand to do Cresyl Violet staining. In this case there is no need for sulfide pretreatment. Just use a Saline solution (0.9% NaCl) plus Heparin to flush the vessels. This is very important because otherwise the vessels will be clotted immediately after you have started with the PFA-perfusion. This results in a bad tissue preservation. You can use this protocol for all Histochemical and immohistochemical reactions as well as for ISH. The rest of your fixation protocol looks good.

Hi Ola Alshaqi

Have you checked if the bubbles are formed immediately after cryosectioning or during staining?

I would suggest you to fix the brain using PBS and 4% PFA (5ml*3 times) and then store the brain in 4 degrees for 24hrs, followed by 15% and 30 % sucrose (using 15 and 30 % would enhance the permeability of sucrose into the tissues compared to using only 30% directly).

Prior to sectioning keep the brain tissues inside cryotome to optimise the temp of the blocks.

Hope it helps,

Thanks,