[Histonet] Storage of tissues in PFA before dehydration andembedding

Tony Henwood AnthonyH <@t> chw.edu.au
Wed Jun 3 18:53:34 CDT 2009


Where did you hear that "storage in PFA for longer than 24 hrs may
adversely affect my tissues"? The beauty of 4% formaldehyde (especially
if it is buffered) is that it does not adversely affect tissues, in fact
it protects them brilliantly from the rigours of subsequent processing.
If it is immunohistochemistry using formalin-labile antigens that you
are worried about then use frozen sections not FFPE sections.

The major cause of poor morphology in FFPE tissues is inadequate
formalin fixation. If you want ethanol fixed tissues (and rotten
morphology) then leave out the formalin fixation step altogether.

AND how do you fix it in 4% polyformaldehyde? Surely when it is in
solution it becomes formaldehyde?


Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead 
Cnr Hawkesbury Road and Hainsworth Street, Westmead 
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Nicholas
David Evans
Sent: Thursday, 4 June 2009 3:59 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Storage of tissues in PFA before dehydration

Dear all,
I am doing some experiments where mouse skin tissue is harvested for
immuno and in situ staining at 2, 4, 6 and 8 days following a treatment.
The usual protocol used in our lab (for other tissues, usually bone) is
to fix overnight in 4% PFA before dehydration and paraffin embedding. As
I value my weekends, I would obviously prefer to do the dehydration
(which we do in ethanol series, an hour in each of 30, 50, 70, 90, 95,
100% ethanol = 6 hours, before overnight in 100%) and embedding of all
samples on the same day. 
I understand that storage in PFA for longer than 24 hrs may adversely
affect my tissues. Is it possible to store for longer in a lower
concentration of PFA, or in another buffer until I am ready to dehydrate
and embed? In short, I would appreciate if anyone could suggest a way to
plan this experiment in the most efficient way. 
With thanks and best wishes
Nick Evans
Dept Surgery
Stanford University 
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