I am hoping some questions and suggestions will help you think in a way to
solve this problem.

I am making the assumption that the negative control you are speaking of is
a No Primary control rather than a negative tissue control.

If you are not doing a No Primary Control I would highly recommend one. 

If you have a no primary control with terrible background then the
background is due to some other reagent or combination of reagents.

You might want to give some thought to some additional controls to pin the
cause of this background down.

Possible causes would be:
1-  secondary antibody concentration
2 - avidin biotin complex concentration
3- chromogen concentration or peroxide concentration

I would not keep my frozen sections at RT for 1-2 hours I would air dry for
1-5 minutes and proceed with fixation. I you need time to collect as we all
do, try keeping slides in -20 or -80 until ready to start or hold in PBS@4C
following fixation.

The difference between frozen and paraffin is that you need to deparaffinize
and rehydrate paraffin embedded tissue prior to staining. Sometimes you need
to do some sort of epitope unmasking but this can be done on frozen tissue
as well.

You did not say what antibody you are working with. If you are very new to
IHC and frozen brain sections I would highly recommend staining for
something like GFAP first and get some confidence using and understanding
the technical aspects of the system.

Hope this is helpful

Cynthia Favara
NIAID/NIH/RML/LPVD
903 South 4th Street
Hamilton, MT 59840
406-363-9317

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-----Original Message-----
From: Anna Elisse Beaudin [mailto:aep10@cornell.edu] 
Sent: Tuesday, December 07, 2004 1:29 PM
To: histonet@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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