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

[Cath Gorrie]

Take two.

Anna,

In general your protocol should work fine. There is nothing that should
cause terrible background, even allowing for inter-user variation.

Some thoughts to consider...

Are you air drying in a clean dust free environment?
Is the "dirt" on the slide away from your section?
How thick are your sections?
Do you gently agitate the sections during washes? - ie place in a jar of
buffer rather than just streaming buffer over the surface.

I agree that the concentrations of your reagents is a possible cause

and I definitely agree that it would be sensible to trial your techniques
with a well defined and robust antibody. GFAP is an ideal choice. This will
give you a better idea of where the source of the problem is.

Regards, Cathy

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Catherine Gorrie
School of Medical Sciences
University of New South Wales
Sydney, NSW

ph: 02 9385 2462
fax: 02 9385 8016
e-mail: c.gorrie <@t> unsw.edu.au
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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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