Re: [Histonet] need advice on labelling primary antibody withfluorophore; also need suggestions for good NF-kB antibody against mouse tissue

From:"Gayle Callis"

Molecular Probes/Invitrogen has kits to label antibodies with Alexa 488 or 
other Alexa fluorophores.  These are excellent, brighter than FITC and 
rhodamines,  and more resistant to fading.  We do double labellling, even 
triple immunofluorescent staining with antibodies from the same species (all 
rat antiMouse) using two biotinylated antibodies with two different Alexa 
fluor labeled Strepavidins, and one purified primary antibody followed by a 
secondary fluorophore conjugate.  For some cells, doing direct IFA on tissue 
sections doesn't work as the fluorophore quenches itself, so it will not 
fluoresce. The antibody will bind the problem is the fluorophore. 
Quenching  is NOT fading or photobleaching but occurs when there are  i.e. 
FITC molecules too close to each other.  Then quenching occurs.  I am not 
sure if this happens with Alexa dyes, but it may.   We cannot see CD4 cells 
detected with a rat antiMouse CD4-FITC conjugate, and if we use this 
primary, we have to detect with an antiFITC antibody.    Quenching may not 
occur with all antibody-fluorophore, but we have occur several CD markers.

 With double and/or triple IFA staining, we do special blocking to ensure 
there is no non specific binding of secondaries to primaries, etc.  Mouse on 
mouse staining will be a bit different, but one should be able to work with 
biotinylated primaries and Strepavidin Alexa dyes plus Strepavidin/biotin 
blocking steps.

I will be happy to discuss this with you privately, if you wish.

Gayle M. Callis
Bozeman MT 59715

----- Original Message ----- 
From: "Jacqui Detmar" 
Sent: Friday, February 01, 2008 12:24 PM
Subject: [Histonet] need advice on labelling primary antibody 
withfluorophore;also need suggestions for good NF-kB antibody against mouse 

Hi all.  Hope you are having a nice afternoon.  I live in Toronto, so
I'm trying to avoid looking out the window at all that snow coming down
.  Anyway, I have a couple of questions to ask those in

1. I am looking for a kit and/or advice that will help me to label
a green fluorophore (possibly fluorescein?) to a primary antibody, that
also comes with it's own anti-"green fluorophore" secondary antibody
(also labeled with a green fluorophore).  I am doing double-labelling
studies using two different primaries from the same host species.  In
the future I will also likely be doing mouse-on-mouse staining and hope
to circumvent high non-specific staining by labeling with a fluorophore.

2. Does anybody have any experience with a good NF-kB antibody for
IHC on FFPE mouse tissues?  I would love to know what works for you.

Thanks all, and enjoy the weekend,


Jacqui Detmar, Post-doctoral Fellow

Samuel Lunenfeld Research Institute, room 876

Mount Sinai Hospital

600 University Avenue

Toronto, ON, Canada

M5G 1X5

phone:   416-586-4800 x2451/x2290

fax:        416-586-8588


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