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

Fri Dec 12 09:16:20 CST 2008

Oh, I'd like to know that, too, please!

--On Friday, December 12, 2008 7:48 AM -0600 "Walters, Katherine S" 
<katherine-walters <@t> uiowa.edu> wrote:

> 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 <@t> 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 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
>
>
>
> ·¢¼þÈË£º Tony Henwood
> ·¢ËÍÊ±¼ä£º 2008-12-12  06:18:47
> ÊÕ¼þÈË£º Pat Flannery; histonet <@t> lists.utsouthwestern.edu
> ³ËÍ£º
> Ö÷Ìâ£º 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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