Re: Whole mount Immunos

From:Dianne Holmes <> (by way of histonet)

Jill, I had a similiar problem when working with rat brain material.
First, rinse sections throughlly  in  PBS before the DAB incubation.  If
residue still appears, sonicating the sections for 30-60 sec will help.  If
background is still dense after the DAB, use a 0.1% buffered glutaraldehyde
(TAAB) solution rinse for 2 - 5 minutes.  This worked for me.  Good luck.

>>> Geoff McAuliffe <mcauliff@UMDNJ.EDU> 09/06/00 07:31AM >>>
Jill McVee wrote:

> Dear Histonetters,
>                   I am currently engaged in staining whole mount frog
> embryo nervous systems for immuno and although I,ve done the usual
> things(diluted AB, blocked for endogenous peroxidase, added things to the
> Normal serum block , diluted the DAB) I am still having background
> problems. Has any one used the method where you take the sample through
> alcohols into solvent, then back again and leave in PBS overnight before
> you start staining if you have please reply and A. tell me if this might
> help the background problems B.give me advice and /or methods which
> will.The last two times I have posted a similar request no one answered so
> someone some where this time please please answer!!!!!!        Thank you in
> anticipation.
> Jill McVee
> Histologist
> St. Andrews Uni
> Scotland.


    I seem to remember the graded alcohol series as a way to increase
permeability to immunoreagents, not reduce background. I can look up a
reference if you like.

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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