Re: Vector Mouse on Mouse Kit

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To:Emma Carter <>,
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     Hi Emma!  I don't have alot of experience with mouse tumor tissue but I
would like to make a few suggestions!  You might consider to incubate the
primary, secondary and enzyme at 4 C instead of RT.  This has helped me when I
do alpha actin staining (monoclonal) on mouse tissue. Another possibility is the
application of a biotinylated primary antibody thus eliminating the secondary
antibody.  I have used this concept with monoclonal PCNA antibodies on mouse
tissue and it works very nicely!
     I did one very brief study with mouse tumor tissue last year and had lot's
of background/non-specific staining when using HRP/DAB.  A few suggestions that
were offered to me were the following:  1)  use 3% H2O2 to block endogenous
peroxidase activity as tumor tissue is highly metabolic (I did this in my frozen
sections with no problems).  You may also consider the alternative peroxidase
blocking protocols that have been discussed over the Histonet recently; more
specifically the glucose oxidase/glucose/sodium azide method.  I personally have
not used this protocol but a co-worker has and was very pleased with the results
in frozen spleen and lymphode tissue.  2)  Use a high salt buffer rinse after
application of the primary, secondary, and enzyme steps to also eliminate
non-specific/background staining.  I incorporated this step as well as
incubating my slides at 4C.  My results were great for CD45 and F4/80 IHC in
frozen mouse tumor tissue.  Interestingly, it was the best F4/80 staining that I
have ever achieved!
     I hope this information is helpful.  Best wishes!


Jennifer Hoover
Novartis Pharmaceuticals

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