I am working with stroke animal models and have to perform the immunohistochemistry time and again. To label the astrocyte I generally used the GFAP marker with 1:500 dilution of the antibody. The problem was GFAP labels the astrocytes only in the edges of cortex and sometime in the layers in between cortex and striatum. It labels the brain slices from the sham operated brain slices as well (where actually it should not). So how can I eliminate this problem of overall staining in brain slices and not in sham operated group?
GFAP will stain all astrocytes in the brain, not just activated astrocytes after injury. You will somehow need to analyse the increase from the background levels seen in the shams.
Agree with Catherine, GFAP will stain all the astrocytes in the brain. So you have a lack of signal that could be related with problems during perfusion (if any) or antibody access to the tissue (free floating vs. slide IHC) just to name two.
Typical GFAP staining in the dentate gyrus of a control animal:
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