[Histonet] Storage of tissues in PFA before dehydration andembedding

[Nicholas David Evans]

Dear Tony and others,

Thanks you very much and to others for the very helpful suggestions. I
suppose there are many different ways to skin a cat!

I will take on board all the valuable suggestions and use them to optimise
my protocol.

With thanks and best wishes
Nick

-----Original Message-----
From: Tony Henwood [mailto:AnthonyH <@t> chw.edu.au] 
Sent: 04 June 2009 16:15
To: Nicholas David Evans; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Storage of tissues in PFA before dehydration
andembedding

Nick,

Well after 30 years of doing ICCs, the worst results are nearly always
with tissues that have not been adequately fixed in formalin. This is
based on using over 300 different antibodies in both adult and pediatric
settings.

The accompanying paper shows an example of what can go wrong with
under-fixed tissue:

Gomes L, mackie N, Catchpoole D, Henwood T (2008) "Test Your Knowledge"
J Histotechnol 31(4):138-184


Regards

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: Nicholas David Evans [mailto:ndevans <@t> stanford.edu]
Sent: Friday, 5 June 2009 8:56 AM
To: Tony Henwood
Subject: RE: [Histonet] Storage of tissues in PFA before dehydration
andembedding


Thanks for the helpful response. 

After a consensus of views I decided to store at 70% ethanol for several
days following 3-4hrs in 4% PFA. I think many people use paraffin
embedded tissues for ICC and over-fixation is deleterious for this. 4%
Paraformaldehyde (PFA) = 4g PFA dissolved in 100 mL PBS. Indeed when in
solution it is present as formaldehyde. I will consider your suggestion
of frozen sections if I have poor results.

Thanks again for the e mail and best wishes
Nick

-----Original Message-----
From: Tony Henwood [mailto:AnthonyH <@t> chw.edu.au] 
Sent: 03 June 2009 16:54
To: Nicholas David Evans; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Storage of tissues in PFA before dehydration
andembedding

Nick,

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?

Regards

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
andembedding


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 _______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

*********************************************************************
This email and any files transmitted with it are confidential and
intended solely for the use of the individual or entity to whom they are
addressed. If you are not the intended recipient, please delete it and
notify the sender.

Views expressed in this message and any attachments are those of the
individual sender, and are not necessarily the views of The Children's
Hospital at Westmead

This note also confirms that this email message has been virus scanned
and although no computer viruses were detected, The Childrens Hospital
at Westmead accepts no liability for any consequential damage resulting
from email containing computer viruses.
**********************************************************************


*********************************************************************
This email and any files transmitted with it are confidential and intended
solely for the use of the individual or entity to whom they are addressed.
If you are not the intended recipient, please delete it and notify the
sender.

Views expressed in this message and any attachments are those of the
individual sender, and are not necessarily the views of The Children's
Hospital at Westmead

This note also confirms that this email message has been
virus scanned and although no computer viruses were detected, The
Childrens Hospital at Westmead accepts no liability for any consequential
damage resulting from email containing computer viruses.
**********************************************************************