Re: Paraformaldehyde and autofluorescence.

IMMUNOLOGY (immunolo@courrier.usherb.ca)
Tue, 1 Apr 1997 23:54:29 -0500

> Dear colleagues,
> I perform immunophenotyping of human lymphocytes by flow cytometry.
>I use 1% paraformaldehyde solution (PFA; Sigma) in PBS for cell fixation.
>The autofluorescence of fixed cell exited at 488nm is increased 6-7 fold
>comparing to nonfixed control.
> 1. Can someone suggest me an alternative PFA source which gives
>minimal autofluorescence?
> 2. Can someone suggest me a post-fixation protocol which reduces
>PFA-fixed cell autofluorescence?
> Thank you.
> Dr. L. Volkov
*********************
We too use 1% paraformaldehyde solution for the same purposes, (Polysciences
Cat#0418, 10% Ultrapure diluted in PBS). One thing though is that the made
up solution should be kept in the dark as with time, there is a colour
change of the solution to a yellowish tinge...this may be contributibng to
your background fluorescence. We change our fixative (make up new batch),
once a month.
Geza Paukovics - Flow Laboratory - AIDS Pathogenesis Research Unit
Macfarlane Burnet Centre for Medical Research
PO Box 254
Fairfield, VIC 3078
AUSTRALIA.
ph. (+61 3) 9282 2132,
FAX (+61 3) 9282 2100
********************
If you have a Molecular Probes catalog, look for a sodium borohydride
reduction treatment that is supposed to be useful for reducing
autofluorescence. I don't have the catalog with me right now but reply
if you can't locate one and I'll get you the details.
Mike King
UF Neuroscience
*********************
Are you preparing the paraformaldehyde from powder? It is important not to
warm the solution over 59 C, otherwise it converts to formaldehyde and will
cause a serious increase in autoflorescence. Powdered para doesn't disolve
well in water even at 59C, so it helps to add some NaOH. I prepare a 2%
solution by adding the paraformaldehyde to 900 ml water, warming to just
under 59C while stirring on a warmer/magnetic stir plate, and when no more
will disolve, adding NaOH drop by drop until completely disolved. I then add
100 ml 10x PBS, and check the pH. A working solution is prepared by diluting
1:1 with 1x PBS. The working solution is stored in the cold and protected
from light, as is the stock solution. The working solution is discarded
after 1 week, while the stock solution can be kept for one or 2 months. I
have not encountered any significant increase in autofluorescence with this
protocol. When fixing a sample in 1% paraformaldehyde, it is important to do
a PBS (without serum) wash before, otherwise the para is used up fixing serum
proteins, and cells are poorly fixed, resulting in deterioration of the
sample and signal to noise ratio. Hope this helps.
Claude
ClaudeC81@aol.com
*****************
Do you have a stream-in-air instrument ? If so a large proportion of your
autofluorescence may be 90 degree light scatter which is increased with
fixation. In our hands paraformaldehyde doesn't give very much
autofluorescence unlike gluteraldehyde.
Jim Watson
jimbo@sanger.ac.uk (Jim Watson)
*******************
Although I haven't looked specifically at autofluorescence of fixed vs.
unfixed cells, I
struggled extensively to first of all prepare 1% paraformaldehyde (from
crystals). Finally I learned from Cytometry Associates of a supplier of
10% paraformaldehyde solution. Its shelflife is very good and all you need
to do is dilute it upon use. The ordering info:

Electron Microscopy Sciences
1-800-523-5874
Cat. #15712-S
EM Grade Formaldehyde, Methanol-Free, 10% Solution
100 mL
$14.00 each

Should you look at this fixative for autofluorescence, could you please let
me know of any problems?
Thanks,

Cindy Gumbs
Associate Scientist II
Centocor, Inc.
USD1#c#USD1.US04#c#GUMBSC@centocor.com
********************
You did not fully give your procedure,but some suggestions are to wait at
least 1 hour after addition of fixative.This time period(or longer)
eliminates the autofluorescence.Also,old fixative that has not been prepared
fresh will give high autofluor.In addition,if you are using the powder form
paraformaldehyde I suggest you switch to the EM grade methanol free
solution.Refer back to Dr.Darzynkiewcz discussion in this e mail bulletin
board for specifics on formaldehyde vs. paraformaldehyde.
Vincent Falco
vincent.falco@es.nemc.org>
**********************
We do a lot of immunophenotyping on lymphocytes and PMNs as well and we
use 1% Formalin in PBS from a formaldehyde stock
solution(37%w/w;Fisher). We have no problems with high levels of
autofluorescence.

Raffi Manoukian
Royal Victoria Hospital
Montreal, Quebec, Canada
E-Mail: Surglab1@is.RVH.McGill.ca
********************
I use Tousimis formaldehyde solution, it comes as 10X10ml vials (cat number
1008A phone 301 881 2450). I've found it pretty good in the autofluorescence
department if you make it up fresh. A post fixation proceedure you can use is
to wash your cells in PBS after fixation, the cells will still be preserved
for
a week or two and increases in autofluorescence are minimal.

Simon Monard
Aaron Diamond Center
New York
Simon_Monard@adarc.org
*********************
You need to buy 10% ultarapure paraformaldehyde from Poly Sciences
Call !-800-555-1212 (thats 800 number information) and ask for the
number for Poly Sciences.

Hope it helps

Barren, Phil
BarrenP@MedImmune.com
**********************
May I suggest a reference which may be of help to you in your
paraformaldehyde fixation/autofluorescence dilemma? Your message
doesn't specify, but it sounds as though you currently pre-fix your cells,
and the time for which this fixation occurs may be the key. Please try to
get a copy of this ref., as it may shed some light:

Babcock, G.F., and Dawes, S.M. 1994. Immunophenotyping Using
Fixed Cells. Methods In Cell Biology Vol. 41 Chapter 5. Academic
Press.

Best of luck,
Susan Dawes
USEPA/Cincinnat
Dawes.Susan@EPAMAIL.EPA.GOV
************************
From my experience the problem is not the fixative but the
presence of the mono- and granulocytes. If I remember right
you can stop it by washing / stopping the reaction by tris.
You might want to contact Dr. J.Lawry for further info

Gerhard.Nebe-von-Caron@unilever.com
***********************
We use Polysciences, Inc. Formaldehyde (Methanol Free), 10% Ultrapure
EM Grade, Cat#04018. We just dilute it 1:10 with PBS+0.1% NaN3.
Polysciences #(215) 343-6484 or (800) 523-2575. Hope this will help.
Nancy Gin - United Hospital- St. Paul, MN
ssni@ix.netcom.com (Vincent Gin)
***********************
You might try making it up fresh, as paraformaldehyde turns into
formaldehyde, which causes more fluorescence. We also use it cold.
Monte Cooper and Deb Berglund
montedeb@montana.campus.mci.net
**********************
Try electron microscopy sciences--their stuff is not bad. Much better than
sigma

ladd is also a good source.

buy the stuff in 16 or 20% solutions packaged in cryules--in an inert
atmosphere--the highest grade they have--dilute with buffer to your desired
concentration--and do not reuse any leftover stuff.

Just my experience.
Paul Mozdziak
Postdoctoral Fellow
University of Wisconsin-Madison
Department of Anatomy
1300 University Avenue
Madison, WI 53705

608-262-5984
FAX 608-262-7306
**********************
I use to do imunocytochemistry to observe cells in confocal microscopy
and the way we use to diminish background after PFA staining is
incubating cells with 75 mM NH4Cl 20 mM glycine in PBS Ca++/Mg++ pH
8.0 for at least 30 min. I don`t know if you can used the same
protocol for cytometer analysis, but my feeling is that it could work
as in microscopic observations.

Patricia Veras, MD, PhD
Laboratory of Pathology and Cellular Biolology
CPqGM - FIOCRUZ
pveras@compos.com.br
**************

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