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

[Cath Gorrie]

on 8/12/04 7:28 AM, Anna Elisse Beaudin wrote.......

> 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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