[Histonet] How to eliminate holes in mouse brain tissue?

[Amy Janes]

Hi,
I am writing to see if I can get some advice on how to eliminate holes I
am seeing in my tissue.
I am using tissue from the mouse olfactory bulb and brain for fos ICC.
Unfortunately I have been unable to see any fos because my tissue is full
of little holes and therefore I have no actual cells. I have determined
that it is not the ICC that is causing the holes because they are present
before staining. So I am assuming I am doing something wrong during
fixation and cryoprotection. I perfuse the animal with 4% para at a rate
of 3.5ml/min and I give the animal around 50mls. I then post fix with 4%
para overnight and then cryoprotect with 30% sucrose until the brain drops
(usually overnight). The brain is then imbedded in OCT and put in the
cryostat at -20C to freeze and cut at 20microns. I typically do not
perfuse with PBS before para but others in my lab have tried perfusing
with and without PBS and have found no difference in tissue quality. Any
suggestions as to how I can fix this?

Thanks,
	Amy