RE: [Histonet] Rat brain cryostat sections

From:"Charles Scouten"

1)  The problem is probably not due to perfusion pressure.  You would need 9 ft of gravity pressure to equal normal blood pressure.  Unless you used a peristaltic pump at very high flow rate, you could not get enough pressure to do any damage.

This sounds like freezing artifact, the "Swiss Cheese" aritfact due to too slow a rate of freezing.
Did you store in 30% sucrose until the brain sank, indicating saturation?  Maybe 3 or 4 days?

What is your definition of quick freeze?  Solid within 2 or 3 seconds is what is necessary. How did you achieve the freeze?  A pedastal on the fast freeze rack of a cryostat is not even close, would cause the sort of problem you have described, unless throughly saturated with sucrose.


Cordially,
Charles W.  Scouten, Ph.D. 
myNeuroLab.com 
5918 Evergreen Blvd. 
St. Louis, MO 63134 
Ph: 314 522 0300 x 342
FAX  314 522 0377 
cwscouten@myneurolab.com 
http://www.myneurolab.com 
 

-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Michiko
Sent: Friday, December 01, 2006 3:26 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Rat brain cryostat sections

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 4C 
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


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