Replies on paraformaldehyde fixatives
T. Scott Thurmond (thurmons@ehsct7.envmed.rochester.edu)
Fri, 09 Aug 1996 10:15:48 -0400
To the list,
=09 First of all I would like to thank all those who responded to m=
y query on=20
paraformaldehyde (pfa) fixatives (posted in April), secondly I apolog=
ize for=20
taking so long to send a summary of the replies. I received 17 indiv=
idual=20
responses, and 10 different recipes for pfa fixative (with a few for =
formalin=20
or formaldehyde fixatives and another touting BD CellFix). I=B9ve de=
cided to=20
make this a long e-mail rather than add an attachment. I hope no one=
=20
objects. =20
=09 Everyone seemed happy with their own formulation and reported go=
od=20
fixation for flow. In general, most stressed the need for using the =
fixative=20
soon after its preparation to reduce the possibility of formic acid=
=20
generation. From what I=B9ve read and comments in the replies, three=
weeks=20
seems to the longest period before preparation of fresh fixative is n=
ecessary=20
(although a least one respondent reported good fixation with 2% pfa k=
ept=20
light-protected at 4C for over a year, and another implied that solut=
ions=20
were good for up to 2 yrs at 4C). The preparation buffers used incl=
uded=20
PBS (both generic and Dulbeccos=B9s) and Isoton. The prepared=20
concentrations ranged from 1% to 4% pfa, with a 1% solution being the=
=20
most common. The concentration used should probably be predicated=
=20
upon the epitope of interest as Matthias pointed out. There were dif=
fering=20
opinions on the pH of the final solution. Two recipes called for a =
pH of=20
7.0 while two others used a pH of 7.4. All reported good results at =
those=20
pH extremes, although one respondent pointed out that FITC is sensiti=
ve to=20
pHs that vary from 7.3. Temperatures for pfa solution preparation als=
o=20
differed. Most heated the buffer with stirring to 60C though others =
used=20
70C. I=B9m not sure that this is a critical point. Complete solubil=
ization of=20
pfa is also an issue. Paula wrote that pfa readily goes into soluti=
on in=20
Isoton. Others using PBS as a diluent added small amounts of either =
NaOH=20
(2 M) or KOH (5 M). Two people just used gradual heating for up to 2=
=20
hours to get most into solution and then filtered the final solution.=
Some=20
used other additives, such as d glucose, to adjust osmolarity of the =
final=20
fixative.
=09By way of summary, it appears that personal preference, more tha=
n=20
anything, dictates the preparation of pfa fixative. The only conditi=
on that=20
everyone unanimously agreed on was that high quality pfa (EM grade)=
=20
should be used. The only other point that a majority agreed on was t=
hat the=20
final fixative solution has a relatively short shelf life (with excep=
tions as=20
noted above). There was also quite a bit of discussion on whether pf=
a is the=20
superior fixative for flow cytometry. Several people reported no=
=20
differences in quality of fixation for formalin (freshly prepared),=
=20
formaldehyde (EM grade) or pfa.
=09 Once again, thank you all for your input.
Scott
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