Hi there,

Yes, you are right. GAD65 and GAD67 staining are really punctate and it's often hard to tell between positive and negative cells (specially within the pyramidal cell layer), although with GAD67 it's easier...

There is a previous post about this in RG (although it's mostly about GAD67):

https://www.researchgate.net/post/does_anyone_have_a_good_protocol_for_Gad65_67_immuno

The best alternative that comes to my mind is to use the GAD65-GFP mice (http://www.informatics.jax.org/allele/MGI:4847069), if it's affordable or you have someone around who could share. Also, you could try ISH for GAD65...

Check these papers for the mouse:

Article Preferential Origin and Layer Destination of GAD65-GFP Corti...

Article Molecular and Electrophysiological Characterization of GFP-E...

Article Sun QQ, Zhang Z, Jiao Y, Zhang C, Szabo G, Erdelyi F. Differ...

Cheers,

J