Dears,
I have a proplem when staining zebrafish embryo with phalloidin.
When the embryos are fixed with 4%PFA and apply the stain, it's working normally.
But if I do dehydration with methanol to store them in -20, the stain doesn't work after rehydration and proper wash with PBST, and the stain become like autoflurescent
Any suggestion please.
Methanol fixation does not go well with F-actin. You may check this thread
https://www.researchgate.net/post/Problems_with_actin_staining_nuclei_stained_and_no_clear_actin_fibers
so you apply the stain before fixing with 4% PFA or after ? (Note: always use fresh PFA)
you do dehydration with 100% methanol ?
Or try this below given protocol
https://bio-protocol.org/pdf/bio-protocol786.pdf
@Biswajoy
Thank you for your reply, it seems that methanol affect the stain
@udhaya
We are following the same, but when we do dehydration the stain doesn't work after rehydration
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