I am working on a dissection fixation protocol for a non model organism and I am using DM1A alpha tubulin as a control to see if immunos work with a given trial of protocol.
I think I got to a point where it is working but I'd like advises from other people. Indeed I do not see any sort of Astral projections. Yet I am aware that tubulin does not have to be with Astral projections. So I am asking does this convince you it is a working stain ?
Look nice and you surely have runned neg controls.
Only i do not understand why in first image you see blue nuclei in dome cells but not in others
in second image i see blue nuclei in almost every cell
Hi Leo,
The staining looks a little off to my eye. What were your fixation conditions? Tubulin immunostaining can be finicky and requires particular fixation conditions for optimal labeling. Depending on whether or not your other antigenic sites are compatible, it's ideal to use either mixed aldehydes (PFA+ Glutaraldehyde) with simultaneous extraction or methanol. PFA alone gives a hazy staining pattern that leaves much to be desired.
Also, some laboratories also recommend adding a calcium chelator into the fixation buffer as calcium induces depolymerization of MTs.
As Dr. Franco suggested, be sure to run neg. controls with your staining. If you'd like a more detailed fixation protocol I'd recommend looking up papers from the labs of Dr. Mark Black (Temple University) or Dr. Peter Baas (Drexel University).
Best,
Anand
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