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

[Anna Elisse Beaudin]

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