tf,
Answers as follows:
Seems obvious that the 4% formaldehyde (made from PFA) was incorrectly made.
What they did, I have no idea nor did I have the time to show them how to make
it up. My advice: "make it from concentrated 38% formaldehyde (formalin)".
You wrote: "I do think most biomedical labs currently are using PFA to prepare
the fixatives!" - I do not think so. A sweeping statement if ever I have heard
one. This s the type of misinformation that we have to deal with. My experience
(over 30 years) indicates that all pathology labs I have worked in as well as
those I have visited, as well as several Pathology Colleges (eg CAP and RCPA)
surveys indicate that MOST (if not all) histopathology labs prepare their 10%
formalin fixative from concentrated formalin not polyformaldehyde as you have
incorrectly stated.
Be careful of misinformation.
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: tf [mailto:ti...@foxmail.com]
Sent: Friday, 12 December 2008 7:35 PM
To: Tony Henwood; Pat Flannery; histo...@lists.utsouthwestern.ed
Subject: (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
prepare 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
water, then add concentrated PB solution.
We here dissolve PFA in concentrated PB solution directly (heat & stir
for 2-3 hours), then adjust pH to 7.4.
We dont have big problem in tissue quaility....except when one want to
cut 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
________________________________
发件人： Tony Henwood
发送时间： 2008-12-12 06:18:47
收件人： Pat Flannery; histonet@lists.utsouthwestern.edu
抄送：
主题： 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-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Pat
Flannery
Sent: Friday, 12 December 2008 3:59 AM
To: histonet@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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