[Histonet] Rat brain cryostat sections

[Michiko]

Hello,

I have problems with rat brain cryostat section: the tissue has many holes.

Here's the extraction procedures.
1:      Intracardiac perfusion with 0.9% NaCl (+liquitamine) for 2-3 min.
2:      Followed by cold 4% paraformaldehyde for approx. 15 min.
3:      Extraction of the brain and post fix in 4% paraformaldehyde at 4°C 
overnight.
4:      Store in PBS + azide.
5:      Incubation in 30 % sucrose.
6:      Rapid freezing.
7:      Cryostat section 20 um.

Question:  Why are there many holes in brain tissue?

1:      If the perfusion pressure was too high, can this break capillaries 
and make holes?
2:      If the sucrose incubation was not sufficient, rapid freezing can 
destroy the tissue?
3:      Or other factors?

Thank you for help in advance.

Michiko K