B-Gal and Methyl Salicylate
Dr S T May
lsrei at csv.warwick.ac.uk
Sat Apr 9 17:22:11 EST 1994
The blue stain produced by X-gal does diffuse in tissue under
a variety of conditions including ethanols, MS/xylene etc..
One thing you can do to reduce/prevent the diffusion (which
can also produce noticably crisper patterns in tissue retained
in PBS or whatever wash you use, if any) is to postfix the tissue
in (for example) paraformaldehyde (or any other cross linking fix).
I used to routinely store x-gal stained nervous tissue in
paraformaldehyde in the fridge until I was ready to d/h, clear and
mount a batch and apart from the tissue becoming spongy and
impractical to redissect I never had any problems - the staining
remained distinct and crisp.
One second point that you might find useful to know however, is that
stains on cleared tissue often appear fainter anyway. I see this as
similar to looking at writing on an overhead (or window), compared
to writing on white paper. Changing the background can help some.
As a (marginally irrelevant) side note...if you have extraneous
pigment in your tissue that you would like to remove..then you can
add an equal volume of 30% hydrogen peroxide to the postfix. This
removes a wide range of pigments (everything in a fly - from melanin
to the eye pigments) and because the x-gal reaction product is
oxidised anyway, does not remove the blue, but can improve the
contrast with the background (I have a ref if you want).
Hope this helps,
(BTW...I believe you about the stain ;)
P.S. I don't want to start a 'my clearing agent is better than
yours war'...but I find that xylene clears faster and (IMHO)
sometimes better than Me Sal or histoclear (and Me Sal smells
so awful too :)
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