[Histonet] RE: Murine CD4 antibody for immunofluorescence staining

[gayle callis]

Dear Robin, 

 

You Wrote: Can anyone recommend a good CD4 antibody for use in localizing
CD4 in frozen mouse tissue?  We are being asked to do dual immunofluorescent
(IHC.  labeling with the CD4 and another antibody in frozen mouse tissue.  I
know

CD4 localization in mouse tissue is difficult and would appreciate any help
or guidance anyone might have to offer.

Robin R. Dean, Ph.D.

Senior Scientist & Study Director

Comparative Biosciences, Inc.

786 Lucerne Dr.

Sunnyvale, CA

(408) 738-8060

 

********** 

We use BD Bioscience  rat antiMouse CD4 (L3T4)  IgG2a isotype.  Your
negative control will be Rat IgG2a.   You can also purchase this monoclonal
antibody from eBioscience, or Serotec.    

 

We have not found working with murine CD4 difficult and have done triple
immunofluroescence staining with this antibody in combination with CD8 and
PNad.  

 

If you are working with two primary antibodies from the same host, eg. rat
ant mouse CD4 and then rat antimouse CD8, you have to do sequential
staining.  

 

You can do the staining two ways.  

 

Rat antiCD4 biotinylated and come back with Strepavidin Alexa dye.  This
gets rid of the secondary antibody but the negative control, Rat IgG 2a has
to be biotinylated too.  When we work with Streptavidin Alexa dyes, we do a
Streptavidin/biotin block from Vector.   

 

OR you can Rat antiCD4 and detect with donkey antiRat Dylight 488, F(ab')2
frag of IgG from Jackson, adsorbed to mouse.  Rat IgG2a is the negative
control

  

We use a special acetone/alcohol fixation on air dried frozen sections and
at the end,  Molecular Probes antifade reagent, ready to use mounting media
to keep fluorophore from fading.  

 

I will be happy to send our protocol under private reply if you want it that
works to make double immunofluorescence staining more efficient and
background free, and will include a photo of the results.     

 

Gayle M. Callis

HTL/HT/MT(ASCP)

Bozeman MT