Hello,

Adding pictures will greatly help the troubleshooting.

For mouse brains, ideally perfuse with 30mL ice-cold PBS followed by 30mL ice-cold PBS-buffered 4% PFA. Post fixation O/N with the same PFA followed by O/N incubation in PBS-buffered 20% sucrose (if you need to keep your sections at -20°C). Epitopes will be differentially accessible depending on the fixative, hence affecting if you need antigen retrieval.

For IHC, we first perfuse animals with 100mM PBS (maintained at room temperature). PBS flow is maintained till liver starts to lose its redness and become almost dirty or off white in color. Then we starts perfusion with 4% PFA solution.

4% paraformaldehyde (PFA) is freshly prepared by taking 4g of PFA and dissolved in 100mM PBS.

And i think if you prepare the solution correctly (especially 4% PFA solution) and PBS perfusion is done perfectly then you are going to observe the best morphology of tissue section without any blurry background.

I hope this will help you to some extent.

Echoing Louis-Charles's answer - Try perfusing with ice cold PBS (instead of the current 37C PBS) until the blood clears. Then perfuse through with cold 4% PFA and put the extracted brain itself in about 5 mL of 4% PFA in the 4C refrigerator and remove after 24h. Not sure how you are cutting the tissue as that would determine any other steps (PBS, sucrose solution, etc). But that should get you on a better path.

Hi Jo-Be... Regarding to your protocol, I think your PFA 4% is correct, but on perfusing, the temperature of your reagents can be critical to maintain the quality of the tissue. I always use ice-cold saline 0.9% first (around 50 ml / animal) and then 4% PFA also ice-cold. Maintain your collected brains embedded in 4% PFA at 4°C for 24h and change the solution for 30% sucrose after that (the brains will float in contact with the sucrose solution).