SPAM-LOW: Re: [Histonet] IHC and IF

[Patsy Ruegg]

I agree with Kim, your cytoplasmic staining with hrp/dab is probably because
your anti mouse detection is reacting to endogenous Mouse IgG in the mouse
tissue you are using.

Patsy

Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net 
www.ihcrg.org

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Kim Merriam
Sent: Thursday, April 15, 2010 5:39 AM
To: Thotakura, Anil Kumar; Histonet
Subject: SPAM-LOW: Re: [Histonet] IHC and IF

Hi Anil,

Perhaps you are having mouse-on-mouse issues with your IHC (DAB) and you are
seeing antibody cross-reactivity with mouse IgGs (you don't mention your IHC
detection method, so I could be wrong).  You are probably not seeing this
cross-reactivity with the IF method, because the labeling is direct.  If
that is the case, I would be more likely to trust what I saw with the
directly-labeled antibody.

If you have labeled the monoclonal antibody with FITC and you want to see it
with a chromagen, you could use an anti-FITC secondary (Invitrogen makes a
nice Rabbit anti-FITC) which you could then detect with an
anti-Rabbit polymer (or whatever you normally use to detect a rabbit
primary).

Good luck,
Kim
 Kim Merriam, MA, HT(ASCP)QIHC
Cambridge, MA 




________________________________
From: "Thotakura, Anil Kumar" <a.thotakura <@t> imperial.ac.uk>
To: "Histonet <@t> lists.utsouthwestern.edu" <Histonet <@t> lists.utsouthwestern.edu>
Sent: Thu, April 15, 2010 7:14:26 AM
Subject: [Histonet] IHC and IF

Dear All,

I have kind of funny problem.

I am doing IHC (substrate reaction suing Dabs reagent) and IF ( direct
labeling of my monoclonal antibody with flurochrome).

I am working on mouse liver frozen sections. When I did IHC the staining
looks like cytoplasm and if I do IF for the same protein I saw nuclear
staining. I am unable to conclude whether it is cytoplasm or nuclei
staining. 

Please advice. The monoclonal antibody I used is not commercially available.
We made it.

Thanks In advance.

Many Thanks,
Anil Kumar.


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