AW: [Histonet] Formalin (from Paraformaldehyde) stability

[Gudrun Lang]

I think, the kind of her test would be of interest. If she dissolves the
mRNA out of the tissue a fixation shouldn't be "too good". As a result a
weak fixative works better for this test. On the other side, when she does
in situ hybridization on the tissue it has to be a "good" fixation.
In John Kiernans book stands regarding Fomal-saline, that is has to be made
a day before use to let depolymerization to take place. Then it can be kept
for several months.
Regarding NBF made with paraformaldehyde it says, that it can be kept also
for several months.
My assumption is, that the older fixative makes its job too good for her
purposes and that could result in mRNA degradation. And in a publication I
read was mentioned, that with methods, where the AAA-tail of the RNA is used
to isolate it, the adhesion of methylol-groups on the Adenin (via
formaldehyd) can disturb it. 
Maybe my suggestion can help.

Gudrun Lang
 
Biomed. Analytikerin
Histolabor
Akh Linz
Krankenhausstr. 9
4020 Linz
+43(0)732/7806-6754
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Von: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von Johnson,
Teri
Gesendet: Freitag, 22. Februar 2008 23:32
An: histonet <@t> lists.utsouthwestern.edu
Betreff: [Histonet] Formalin (from Paraformaldehyde) stability

I had a question today from a researcher asking about the stability of a
formalin solution made from Paraformaldehyde. She indicated that on one
of her samples, freshly prepared fixative worked well, while one day old
fixative (same batch) did not fix properly. She assures me all
pre-fixative steps were the same. The samples were fixed for 2 hours. I
have no details about her experiments or what she was testing (I suspect
mRNA targets). 

I realize that without the addition of methanol, the solution is not as
stable would would re-polymerize with time. I'm wondering if the
fixative started re-polymerizing and that made the difference, but one
day? Is it possible for one day to make the difference between a
positive and negative result? 

How stable/instable is methylene glycol?

I'm also thinking that by adding methanol (or using purchased 100%
formalin (37% formaldehyde)) would be useful for mRNA targets, since
aldehydes tend to fix nucleic acids poorly.

Why do all my most difficult questions arise on Friday afternoon?

Thanks for any insight you can give me.


Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110

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