[Histonet] Storage of tissues in PFA before dehydrationandembedding

Sun Jun 7 11:10:27 CDT 2009

I agree with Tony, the problem is usually underfixation not over with
aldehyde fixatives, if you do not stabilize the proteins by fixing (Bryan
Hewlett says this takes 24 hrs. no matter the size of the tissue) paraffin
processing will adversely affect your tissue and in some cases the proteins
of interest will be washed away and lost with no change or no amount of AR
to get them back.  Researchers are stuck in the days before AR techniques
when cross linking of proteins by aldehyde fixation was a problem because we
did not know how to retrieve them, but now we do, and the bigger problem is
losing antigens from paraffin processing because they have not been
adequately fixed to protect them.  I recommend 24-72 hour fixation in
aldehyde fixative and then the tissues can be placed in 70% alcohol.

Patsy

Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net 
www.ihcrg.org

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Tony Henwood
Sent: Thursday, June 04, 2009 5:22 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Storage of tissues in PFA before
dehydrationandembedding

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 following 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 _______________________________________________
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