[Histonet] (reply) silly questions.---PFA

Fri Dec 12 07:48:18 CST 2008

This may be another silly question, but how does one test the concentration=
 of formaldehyde in solution?

Thanks,
Kathy

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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces@=
lists.utsouthwestern.edu] On Behalf Of tf
Sent: Friday, December 12, 2008 2:35 AM
To: Tony Henwood; Pat Flannery; histonet <@t> lists.utsouthwestern.ed
Subject: [Histonet] (reply) silly questions.---PFA

"I looked at the sections and the cell shrinkage (and prominent spaces
between cells and connective tissue) indicated that most of the
"fixation" seemed to have occured in the processing ethanols. I asked
him for some of the fixative he used, tested the formaldehyde
concentration and found it to be less than 0.5%!!"

Tony: Do you think this is because of inproper preparation of PFA in his =
lab, or the common problem in all researchers using PFA?
         I do think most biomedical labs currently are using PFA to prepa=
re the fixatives!

So, anyone has the idea on a correction preparation procedure of 4% PFA?
I noticed some of you dissolve PFA powder in NaOH-conditioned alkaline wa=
ter, then add concentrated PB solution.
We here dissolve PFA in concentrated PB solution directly (heat & stir fo=
r 2-3 hours), then adjust pH to 7.4.

We dont have big problem in tissue quaility....except when one want to cu=
t the brain in a cryostat rather sliding microtome.
Many times the brain sections from the cryostat have "cheese" like holes/=
cavities, which almost never appear on sliding microtome-prepared sections.

2008-12-12 

tf 

=B7=A2=BC=FE=C8=CB=A3=BA Tony Henwood 
=B7=A2=CB=CD=CA=B1=BC=E4=A3=BA 2008-12-12  06:18:47 
=CA=D5=BC=FE=C8=CB=A3=BA Pat Flannery; histonet <@t> lists.utsouthwestern.edu 
=B3=AD=CB=CD=A3=BA 
=D6=F7=CC=E2=A3=BA RE: [Histonet] Silly Question? 

Pat,
I agree with you.
In a routine diagnostic histopathology laboratory, it makes little
difference what you use. Around the world for over 100 years most labs
use 10% neutral buffered formalin made from concentrated 38%(or there
abouts) formalin (or formaldehyde).
Researchers, though, are a different kettle of fish. They will tend to
hang on to misinformed, "mystical" methods believing they are being
scientific. Funny, you would think that they, as a group, would be the
ones pushing the boundaries and critically assessing each step of their
research, ensuring that they understand what and why they are doing it.
(Disclaimer - not all researchers are like this, thank heavens!!)
Using a formaldehyde solution made from polyformaldehyde can cause
problems. One researcher used it and wondered why their morphology was
sub-optimal and their p53 immunohistochemistry was negative. He assured
me that he had fixed small samples of tissue for 6 hours in 4%
formaldehyde and then processed them using ethanol, xylene and wax.
I looked at the sections and the cell shrinkage (and prominent spaces
between cells and connective tissue) indicated that most of the
"fixation" seemed to have occured in the processing ethanols. I asked
him for some of the fixative he used, tested the formaldehyde
concentration and found it to be less than 0.5%!!
This also explains the loss of p53 staining. I gave him some of our
routine 10% phosphate buffered fomalin, asked him to fix overnight, and
try agin. Low and behold problem solved.
How's that for a Friday Flamming!!!
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 Pat
Flannery
Sent: Friday, 12 December 2008 3:59 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Silly Question?
Please humor me on this if it's obvious (to everyone but me):  why do  
we use paraformaldehyde (which is so inconvenient to make up) rather  
than buffered formalin or just diluted formaldehyde itself?
It seems that around here, some folks prefer paraformaldehyde (either  
2% or 4%) and others use formalin, while some others stick to diluted  
formaldehyde (I see all 4 on labels for specimens submitted for  
histology).  Is it mostly a matter of personal preference or where you  
were trained (i.e. force of habit) or is there a valid reason to use  
each solution (basically the same chemical once in solution, merely  
buffered or not)?  The only answer I've gotten when I've asked is,  
"That's what we always use."
Thanks.
-Pat Flannery (not a "real" histologist - I just play one in the lab)
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