[Histonet] RE: double stainings

From:Luis Chiriboga

I absolutely agree Chris, and I stopped using NBT-BCIP and have switched to
Fast Red! I was wondering, do you know of any experimental rather than
empirical evidence for the shielding effect? Empirically speaking, alk phosp
may be more sensitive (I believe it has a lower detection limit than
DAB.....based on western blotting?)than peroxidase/DAB, but the perox/DAB
combo produces a superior signal in FF-PE. I have only limited experience
with the x-gal detection systems.

-----Original Message-----
From: C.M. van der Loos [mailto:c.m.vanderloos@amc.uva.nl]
Sent: Thursday, September 23, 2004 6:46 AM
To: histonet@lists.utsouthwestern.edu
Cc: Luis.Chiriboga@med.nyu.edu; Inga.Hansson@neuro.uu.se
Subject: Re: double stainings

Dear Inga and Luis,
The approach of staining the 1st antigen including enzyme visualization,
antigen retrieval and than the 2nd, would be also my advise in this case.
However, when using DAB as chromogen for the first staining sequence please
realize that this enzymatic product may shelter your 2nd antigen very
effectively! This sheltering is not solved by the antigen retrieval step.
Instead of DAB also Dako Permanent Red (Alk. phosp.) or X-gal
(b-galactosidase) survives the antigen retrieval step quite well, whereas
those chromogens doesn't have this sheltering characteristic.
Hope this helps.

Chris van der Loos, PhD
Dept. of Pathology
Academical Medical Center
Amsterdam - The Netherlands

----- Original Message -----
>From  Luis Chiriboga 
Date  Wed, 22 Sep 2004 08:41:04 -0400
To  Joanne Mauger ,
histonet@lists.utsouthwestern.edu, Inga.Hansson@neuro.uu.se
Subject  RE: [Histonet] double stainings
I have used Joanne's idea before, and it has worked quite well. the only
problem I have observed is that the first chromogen sometimes looses it's
"crispiness". So I would suggest that you be careful which enzyme/chromogen
combination you use first. In my experience I have found that peroxidase/DAB
is the least affected while alk-phosp/nbt-bcip is the worse.

-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[histonet-bounces@lists.utsouthwestern.edu]On Behalf Of Joanne
Sent: Wednesday, September 22, 2004 8:03 AM
To: histonet@lists.utsouthwestern.edu; Inga.Hansson@neuro.uu.se
Subject: Re: [Histonet] double stainings

Dear Inga,
For double staining such markers, you can first stain the antibody that does
not require heat. After the chromogen step, do your heat retrieval, and then
stain with the next primary Ab.

>>> "Inga Hansson"  09/22/04 07:05AM >>>
Hi everyone!

If I want to do a double staining using one antibody that requires
HIER and another that will stain everything if heated........what do
I do?
Can anyone help me?

Thanks !


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