Re: Whole mount Immunos

From:"L. Gibbs" <> (by way of histonet)

Hi Jill-

I will send along my DAB formula in hopes that it could be
helpful. For floating sections I use this diluted DAB formula:
DAB at 0.037% and H2O2 at 0.015%. I prepare this from stock DAB-
Sigma# D5637 /5 grams DAB to 132 ml 0.05M Tris pH 7.6. I aliquot
at 2mls and 4mls.
DAB for floating sections:
2 mls stock DAB
200 mls 0.05M Tris pH 7.6

I do a pre-incubation for 10 minutes at 18C with 100 mls of the
DAB solution sans H2O2. To the remaining 100 mls of DAB solution
I add 0.5ml 3% H2O2 (can be prepared from 30%). I incubate the
sections at apx 18C until there is appropriate color development.

One other idea...if your antigen and tissue can take it, you
could try the sodium borohydride treatment which precedes the
primary antibody. This treatment is harsh but it does bleach the
sections and provides nice contrast for the chromagen.

Good luck!

Lorraine Gibbs

On Wed, 6 Sep 2000, Dianne Holmes wrote:

> 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.
> Jill:
>     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
> --
> **********************************************
> 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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