[Histonet] IHC on brain cryosections... help!!

[Charles Scouten]

I agree with Geoff McAuliffe in every detail, you need to perfuse, and you need to fast freeze.  If perfusion is new to you, I suggest you review the manual and protocol posted on the left at the following link.

http://www.myneurolab.com/myneurolab/mnl_products_detail.asp?idproduct=471001&catdesc=Histology+Equipment&CatThreeID=674&CatOneID=4&subcatdesc=Sacrifice+Equipment&idsubcategory=21

About the need for fast freezing, see

http://www.myneurolab.com/global/Manuals/Tips%20and%20Techniques%20Freezing%20Artifact.pdf




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


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Anna Elisse Beaudin
Sent: Tuesday, December 07, 2004 2:29 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] IHC on brain cryosections... help!!

Hi all,

   I am having a TON of trouble with basic IHC on mouse brain cryostat sections!  I have reviewed tons of protocols from the web and from diferent papers.. and I am constantly getting confused between protocols for fixed, paraffin-embedded sections, and those for
cryosections.    Here is an overview of the protocol I am currently
using:

-Collect cryosections on slides, leave at room temp 1-2 hours (as I'm
collecting)

- Quick-fix in buffered PFA (4%) for 10 minutes
- Rinse 3-10' PBS
- Block for endogenous peroxidase activity in 3% hydrogen peroxide in PBS
- Rinse 3 * 10 min Tris-buffered saline
- Block in 1%BSA, .3% triton X in TBS
- in primary o/n at 4 C
- Rinse 3 * 10 min TBS
- in secondary 1 hr at room temp
- Rinse 3* 10 min TBS
-- avitin-biotin (vector elite kit) for half hour at room temp
-- Rinse 2 * 10 min TBS, 1*10 min TB pH7.6
-- in DAB 2-3 minutes

With this protocol, I get terrible background in my negative controls, and cannot identify any stain  in my positive slides.  The background I'm getting does not appear to be cellular at all -- there is just brown gunk everywhere.  Can anyone point me in the right direction?  Thank you so much in advance for your help!

Best,
Anna Beaudin
Division of Nutritional Sciences
Cornell University
Ithaca, NY



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