Hello,

did you also stain other neural tissues, e.g. a slice from the forebrain with cortex, striatum and corpus callosum? How did this look like (perhaps it is a special phenomenon, only happening in the spinal cord)?

Did you use TritonX100 or Tween20 for tissue permeabilization, to get the anti-GFAP into the astrocytes? Iba1 is an epitope on the cell surface and therefore this staining worked perhaps much better without using TrionX100.

I have similar problems when I use a polyclonal Anti-GFAP (rabbit) from Sigma, in comparison to a monoclonal Anti-GFAP (mouse) also from Sigma, thereby less regions are stained for GFAP. I have only a positive staining in Cortex and corpus callsoum, whereas in the striatum there is only a weak staining.

Kind regards

Stefan

Hello Stefan,

Thank you so much for your reply.

Yes i used Tween 20, in all the buffers that i used. And i havent tried to stain other regions. apart from L4/L5 spinal cord segments. I think i should follow your advice and see.

GFAP staining is very straight forward. Most of the commercial antibodies should work even minor protocol changes. Whose antibody you are using?

Instead of NGS try NDS blocking. May be longer primary incubation help.

Hello Govindaiah,

Thank you so much, i will try and use NDS, or increase the incubation time. though that means finding a secondary antibody hosted in a donkey. I incubate over night.

I am using an antibody from MIllipore. MAB360 Ms GFAP.