Perfusion will definitely help.
Using controls is absolutely essential in any experiment and will help determine what is false positive but it will not show false ˇčnegativesˇč. If you are staining a bloody tissue like unperfused heart etc the blood IgG will bind to you secondary abs and it is possible that you will not be able to detect you antigen or it will be weakly stained. Get rid of that background and the positive stain would 'pop'.
Using dapi staining is fine, you should probably be doing this anyway, but the problem of poor staining quality still remains (unless your tissues are well-perfused).
I would consider to do this only in the most desperate of cases.
Sent: Jul 16, 2008 10:35 AM
Subject: [Histonet] Rat-source primary antibody on Rat tissue
I am writing to discuss the use of rat-sourced primary antibody on rat tissue.
One concern (from Andrea Hooper's last email to me) is that the 10mg/ml IgG in normal blood will result in great background, or false-positive staining.
How do you think?
My personal opinions are:
(1) perfuse the rat well.
(2) use a control group without 1st antibody, just add goat-anti-Rat IgG, for example (care about the cross reactivity)
(3) use DAPI staining to see if the staings are located in cell, rather by blood contamination.
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