[Histonet] Murine CD4 and CD8 revisited

From:Gayle Callis

Dear All, 

You wrote: 

I am trying (for a long time all ready) to produce a good CD4/CD8 staining
of frozen stomach tissue (mice). We use the Dako rat
anti-mouse CD4 antibody but always encounter a large amound of background
after staining (it seems like there is a lot of cross
reactivity with the glandular epithelium). Can anyone help me to solve this
problem ?
Thanx !

You did not say how you do your actual staining, and if you use some of
DAkO's kits with mutiple links secondaries, beware, they may be cross
reacting with the mouse tissue if it antimouse included. We have a purist
attitude in our lab, and do hundreds of murine IHC protocols a year, with a
lot of CD4 and CD8 included, even double immunofluorescent staining with
these two antibodies.   

has been reported on Histonet innumerable times as there is NO (yes, I am
shouting!) retrieval of digestion method that will unmask those antigenic
epitopes after formalin fixation.  Sorry folks, just the mousie facts so
don't waste your time.    

We never get background with our Murine CD4 or CD8.  Our antibodies are
monoclonals, rat antiMouse from Pharmingen, that is the first important
step is to purchase the correct primary antibody. 

We work with either pure primary antibody or biotinylated primary and have
equally good results.  And with our biotinylated primary, coming back with
SA-HRP, the dilution has gone out to 1:15,000 (0.5mg/ml).

Frozen sections are air dried overnight at RT, then fixed with 75%
acetone/25% absolute ethanol for 5 min at RT, then immediate rinses with
DPBS. No substitution on alcohol, methanol is avoided at all costs.   
Preferred endog peroxidase block is glucose oxidase method. 
Normal serum block is 10% goat/2.5% mouse serum in Dulbeccos PBS, with
0.05% Tween 20
Use Strepavidin/biotin block from Vector rather than avidin/biotin block. 
Purified antibody is diluted in 1 - 5% goat, 30 min at RT  You would have
to do a dilution panel to determine optimal working concentration. 
Secondary antibody is Goat antiRat F(ab')2 frag of IgG adsorbed to mouse,
so you done get cross reaction to fc receptors on tissue.  1:250 (0.5mg/ml)
 from Biosource/TAGO, 30 min RT
SA-HRP 1:500 (0.5 mg/ml) from Biosource, 20 min
AEC+ from DAKO - we control with microscope but usually around 2 - 5 min. 

We more often use Biotin conjugated rat antiMOuse CD4 or CD8 diluted in the
NSB with 10% goat/2.5T mouse serum, same incubation time
SA-HRP same as above
AEC+ (primary is diluted 1:500 with this, could probably be less
concentrated in your hands). 
If you use DAB+ from DAKO plus their enhancer, this is more sensitive and
you can get that wonderful dilution out 1:15,000!  Have pictures to prove
it, and there is NO BACKGROUND. 

Use normal spleen as a control, the negative control is Isotype matched and
with biotin-primary, the negative control must be biotinylated isotype
matched IgG. 

We NEVER use whole IgG secondary antibodies with mouse, always F(ab')2
fragments, and Jackson's Donkey antiRat F(ab')2 fragment will work, but you
must change your normal serum block to donkey rather than goat with 2.5%
mouse serum.  YOUR secondary MUST be adsorbed to mouse.  We have also used
mouse antiRat F(ab')2 fragment (Jackson) , but you must add Rat IgG to the
diluent of the primary.  Don't recall mg/ml on that, but could look it up,
the results were still clean, no background here either.  

If you need a protocol, I will be happy to file attach privately. Also the
glucose oxidase peroxidase blocking method.   

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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