[Histonet] commercial antibody diluents w/ surfactants

[Brianna Mbow]

Yes, I am planning on doing immunofluorescence staining and would like
to adapt a protocol to the autostainer, which has the built in washing
fluid.
I previously did fluorescence staining on the bench, and I know I always
used triton-x for permeabilization.  I'm now just wondering if I begin
with
trying the commercial antibody diluent (in keeping with the general
practice of the lab), is the surfactant already in the solution enough
for 
permeabilization or would I need added triton-x?  And if I would need to
add more, why is this not necessary with immunohistochemical staining? 

Thanks again,
Brianna Mbow
Antibody Technologies
Seattle Genetics
Seattle, WA

-----Original Message-----
From: Gayle Callis [mailto:gayle.callis <@t> bresnan.net] 
Sent: Friday, April 18, 2008 11:11 AM
To: Brianna Mbow; histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] commercial antibody diluents w/ surfactants

Question:  are you planning to do immunofluorescent staining even though
you 
are now doing only enzyme immunohistochemistry with these reagents?

The size of the fluorophore molecules is not a factor but there are some

other considerations when doing IF.

Gayle M. Callis
HTL/HT/MT(ASCP0
Bozeman MT 59715



----- Original Message ----- 
From: "Brianna Mbow" <bmbow <@t> seagen.com>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Friday, April 18, 2008 11:47 AM
Subject: [Histonet] commercial antibody diluents w/ surfactants


Hello,

In my previous position, I prepared all my own antibody solutions for
fluorescent staining myself with varying levels of blocking serum and 
triton-x for
permeabilization.

I'm now working in a lab that uses commercial antibody diluents and an 
autostainer with its
own washing solution.  Both the diluent and wash solution contain 
surfactant, but the concentration is
not listed.  I'm just wondering if anyone knows if the amount is
sufficient 
for permeabilization in
fluorescent staining.  So far in the lab we have only really been doing
IHC 
and they have never added triton-x to
their solutions.  Also, out of general curiosity, is there a reason for 
needing more permeabilization in IF
compared to IHC staining?  Is it because of the size of fluorophore 
molecules?  Any ideas would be much
appreciated.



Thank you,

Brianna Mbow

Antibody Technologies

Seattle Genetics

Seattle, WA



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