Author Topic: Adivce on IHC Staining pattern needed!  (Read 450 times)

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Offline EJosephine

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Adivce on IHC Staining pattern needed!
« on: March 31, 2018, 05:24:34 AM »
Hello,

I am in desperate need of advice on how to best to interpret/describe the pattern of staining I have achieved through IHC staining on mouse brain tissue. Unfortunately I have no further time to further optimise the protocol and have got to write up and discuss what I am seeing for an important final year project.

I have been staining mouse brain 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 primary antibodies, including my supposed negative control (reported to react with human and not mouse tissue.)
I have attached a link to some of the images from these experiments:  https://photos.app.goo.gl/Ahw2W8nqyxUn0u4E3 (description of image is in the info for each picture)
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.
Thanks!

Adivce on IHC Staining pattern needed!
« on: March 31, 2018, 05:24:34 AM »