Emily, thanks for the input. I've also followed the protocol of
Schaeren-Weimers and Gerfin-Moser's as well and it worked pretty good
for me (14 um frozen section of adult and embryonic brains). However, I
came across with this Maleic acid protocol when I read a recent paper
from Oscar Marin's lab (J. Neurosci, 2007, 27, 9682-9695). Then I went
back and checked the data sheet coming with the anti-DIG-AP antibody
(Roche), I found that they suggest using Maleic acid based buffer for
"membrane application" and Tris-based buffer for "other application".
And a round of Google search of Maleic acid buffer turned up tons of
protocols using maleic acid based buffer for all kinds of in situ,
either whole mount, frozen or paraffin sections. So I am curious why
maleic acid would be preferred in other protocols, as it has much less
buffering capacity than Tris in the pH range of 7-8. Google search
revealed one explanation that maleic acid buffer can be treated with
DEPC-H2O, while Tris buffer can't. The explanation makes sense, but for
posthybridization steps in situ, DEPC-treated buffers are not necessary.
So is there another reason for the preference of maleic acid?
You are right by saying "if it ain't broke, don't fix it", and I am
going to stick with my old protocol. But, still, a little curiosity
hangs over here.
Chengkang Zhang Ph.D.
Room 357, MedSurge II
Department of Pharmacology
University of California, Irvine
Irvine, CA 92697-4625
Tel: (949)-824-1902 (lab)
Emily Sours wrote:
> I've never heard of using maleic acid for DIG in situ's. This could
> be because we have followed Schaeren-Wiemers and Gerfin-Moser (1993)
> protocol, which uses Tris-HCl and makes no mention of maleic acid.
> In the papers I've read using DIG in situ's (with chick embryonic
> tissue, mind you), there has been no mention of maleic acid. Where
> have you seen this in a protocol?
> I suggest "if it ain't broke, don't fix it". When we've tried to make
> our in situ's better by using someone else's protocol, we've *always*
> gone back to our original protocol because it's much better. After
> ten years of using it, we know it kicks ass. Well, at least I do--my
> PI has doubts, but she comes around on day three. :)
> By the way, is this for whole mount or section in situ's? And if for
> sections, for paraffin or frozen, at what thickness? I'm interested in
> knowing in case this helps for paraffin in situ's, since we had
> beautiful results and then nothing (though the embryos were fixed in
> toluene, which didn't work, instead of xylene, which worked).
> Paraffin in situ's would rock my world.
> yes, paraffin in situ's rock my world. because i love my job.
> Yog-Sothoth knows the gate.
> Yog-Sothoth is the gate.
> Yog-Sothoth is the key and guardian of the gate. Past, present,
> future, all are one in Yog-Sothoth.
> He knows where the Old Ones broke through of old, and where They shall
> break through again.
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