you don't mention what fixing solution/ duration you use......
I have not used the primary Ab you mention: however, as it is not a
neurone-specific Ab, you need to use another neurone-specific Ab to confirm
that what you are demonstrating are neurones ( sure, morphology helps but is
not neccessarily definitive) OR switch to a neurone-specific beta tubulin
( beta III tubulin- neurone specific).
Otherwise , it seems to me that your technique is about right, provided that
you are permeabilising after fixation but before primary Ab incubation. You
will need to permeabilise to optimise staining, dependant on all the other
variables in your technique, that you haven't mentioned
NB: Can't comment on appropriate dilution of your primary Ab but your
Alexafluor 488 should be fine......altho' I use it at 1/1000 dilution
Have a look at IF pics of neurone -specific beta tubulin here, for example :
Best of luck
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