I have been staining mouse tissue using primary antibodies raised in mouse, trying to detect microglia using an anti-CD45 antibody (the best marker we had available) and a membrane-bound protein EpHA1 (the expression of which is not yet defined in the brain.)
To try and overcome the problems of my antibodies interacting with endogenous mouse Ig, I have used a M.O.M detection kit, which utilised an ABC detection method. I have used Bloxall to block endogenous enzyme activity, alongside avidin/biotin blocking.
I have obtained a similar pattern of staining for all of my antibodies using, including my supposed negative control (reported to react with human and not mouse tissue.)
I have run out of time for additional optimisation of the protocol and now have got to write up and the discuss the results I have got. However I am struggling with how best to describe/interpret what I am seeing. I have attached a document containing some of the images taken.
Would this be best described as "non-specific, diffuse staining?" seeing as all antibodies are eliciting a similar picture? The "blank" slides incubated with the secondary antibody only show a cleaner picture, but still have some background staining.
Any input would be much appreciated!
I think you should avoid using mouse rised primary antibodies to detect mouse tissue in IHC, then you might see something else.
With the condition that you applied the same exposure settings on the microscope, you can quantify the expression using ImageJ software (NIH), and present the results in comparison to the normal untreated brain.
I would suggest that you are seeing non specific staining of your secondary (no primary control) as well as non-specific primary antibody staining since you have so much background staining in addition to the no primary control staining.
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