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

From:Geoff McAuliffe

Hi Anna:

    Allowing unfixed sections to dry for two hours at room temp. is 
going to give you terrible morphology. Furthermore, loss of cellular 
integrity will allow the antigens you are looking for to diffuse out of 
their normal location, compounding your troubles. Solution? Fix by 
perfusion of the whole animal first with formalin/paraformaldehyde, then 
rinse out the fix, cryoprotect with sucrose, freeze and section. Then do 
your IHC.
    Also, you did not say how you are freezing the tissue. Tissue must 
be frozen very quickly for good morphology, putting it in the cryostat 
and waiting for it to freeze will be a disaster. If that is what you are 
doing, everything else is pointless.
    I suggest you try your IHC on well-fixed material first to see what 
good morphology and good localization looks like. Then move on to 
cryosections. Find someone in the Biology Dept./Vet School/Med School 
who has experience and spend an afternoon in their lab. Seeing how it is 
done will save you time in the long run.


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
>-Collect cryosections on slides, leave at room temp 1-2 hours (as I'm
>- 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!
>Anna Beaudin
>Division of Nutritional Sciences
>Cornell University
>Ithaca, NY
>Histonet mailing list

Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

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