V-CAM/ICAM staining in mouse

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From:Jamie Erickson <JErickson@genetics.com>
Date:Thu, 29 Jul 1999 15:12:50 -0400

Good Morning all,
        I am having a lot of trouble with my I-cam & V-cam staining lately  and was wondering if people have any solutions. I've been trying to stain for I-cam and V-cam in this mouse model of angiogenesis. This model uses a mixture of tumor cells and a material called matrigel that is injected under the skin. This matrigel forms a solid plug under the skin when injected into the mouse (it's hard at 98 degrees and semi-liquid at or below 4 degrees C ). My antibodies only work with frozens so I cryosectioned the plugs with the Tape Transfer system from Instrumedics, the sections look great. 
        Now to the problem, my antibodies seem to be staining everything (Skin, Glands, Adipose tissue, Vasculature and matrigel) I tried diluting the antibody out to 1:25,000 (20 ng/ml)  it doesn't dilute out and the Rat IgG2a and Hamster IgG look the same as the V-cam & I-cam respectively.  Here is what I do : I use a Ventanna Tech Mate 500 Auto stainer for the staining,  I Fix the slides for 10 minutes prior to staining with Acetone and block with Avidin/Biotin block (Zymed) for 10 minutes each. The autostainer  blocks with a block they sell and I supplement 5% horse and 5% goat serum. it uses 3% H2O2 as a peroxidase block total time: 8 minutes. The Primary antibody time is 1 hour, secondary 25 minutes the Chromagen is NovaRed ; 3 times 1 minute each (wash in between steps). I get very nice red staining everywhere, it is not specific but I can figure out what is the cause, any Ideas? 

Jamie Erickson
Genetics Institute
Andover, MA


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