>Date: Thu, 07 Sep 2000 08:41:33 -0700 (PDT)
>From: "L. Gibbs" <lgibbs@u.washington.edu>
>Subject: Re: Whole mount Immunos
>To: Dianne Holmes <dholmes@anatomy.umsmed.edu>
>Cc: histonet@pathology.swmed.edu, jm36@st-andrews.ac.uk, mcauliff@UMDNJ.EDU
>
>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
>UW
>
>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
>> mcauliff@umdnj.edu
>> **********************************************
>>
>>
>>
>>
>>
>>
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