[Histonet] Rat brain cryostat sections
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
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.
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