RE: [Histonet] Rat brain cryostat sections

From:"Charles Scouten"

Yes, when metabolism stops, and the sodium and other pumps shut off, sodium runs in to the cell down the gradient, even if isotonic saline is used, and the interior of the cell becomes hypertonic.  Water must enter to balance osmolarity.  That is why cells swell in response to almost any challenge that shuts down metabolism. I have discussed this in an article in the May 2006 issue of Microscopy Today.

Soaking in 30% (very hypertonic) sucrose should have "dehydrated" those cells, though.  

Yes, they might have already burst if saline was continued for too long before fixative arrived.

When you find what works, please let us know.  The solution will reveal the cause.


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: Tony Henwood [mailto:AnthonyH@chw.edu.au] 
Sent: Wednesday, December 06, 2006 4:19 PM
To: Charles Scouten; Michiko; histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Rat brain cryostat sections

My experience though indicates that slow freezing my not be the culprit.
Normal Saline seems to perfuse quickly into the tissue causing excessive ingress of water into cells. Normal Saline, despite what one would think, is not osmotically neutral with cells.
We had the same problem with brain tissue for frozen section. When tissue arrived in saline, large freezing like holes appeared. When tissue was received on moistened card, the artifact disappeared.

Use a culture medium if you can and the chance of this freezing artifact (or osmotic artifact) may indeed go away.

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) Laboratory Manager & Senior Scientist The Children's Hospital at Westmead, Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318




-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Charles Scouten
Sent: Wednesday, 6 December 2006 4:52 AM
To: Michiko; histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Rat brain cryostat sections


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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