Re: Help with CD 45

From:Gayle Callis <>

What is the other antibody?  Rodent CD markers often do not work well with
ANY formalin fixatives even when retrieval/digestions are used. There are
other fixatives that may work better or use frozen sections (See Pharmigen
catalog for a zinc TRIS buffer fixative for paraffin sections that
eliminates antigen retrieval.  One can also try paraformaldehyde fixation. 

Found this in my files:  CD45R (approx 2.5 ug/ml), digested with proteinase
K (25 ug/ml) 15 min at 37C.  Try a more sensitive chromogen, DAKO DAB+ with
their new enhancer to improve detection if you have a hint of staining.
Some prefer enzyme digestion (trypsin, pronase) instead of buffer antigen
retrievals and just one buffer retrieval may not work, you might have to
try different pH ranges (kits available commercially).  

If you use methanol/hydrogen peroxide to block endog peroxidase, methanol
can (info given out by vendors, interfere with CD marker staining.  Some
have experienced it first hand and work with H2O2 in PBS or TBS, 0.3% H2O2
for 20 min.  Have even added 0.1% azide to this, some prefer to not use
azide or even increase H2O2 concentration, 0.5% etc.

Overnight antibody incubation with formalin fixation may improve staining. 

Good luck

At 11:35 AM 11/30/00 -0500, you wrote:
>Hello Histonetters,
>Would anyone like to share their protocols for doing CD 45 on both Zinc
>Formalin fixed and 10% Formalin fixed Rat tissues.
>I have been having a hard time getting 2 different antibodies to work.
>Any help would be greatly appreciated.  Thanks in advance.
>Jean Reed
>MCD Pharmacology Dept.
>Novartis Pharmaceuticals Corp.
>Summit, NJ 07920
Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303

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