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First of all I would like to thank all those who responded to my query on
paraformaldehyde (pfa) fixatives (posted in April), secondly I apologize for
taking so long to send a summary of the replies. I received 17 individual
responses, and 10 different recipes for pfa fixative (with a few for formalin
or formaldehyde fixatives and another touting BD CellFix). I've decided to
make this a long e-mail rather than add an attachment. I hope no one
objects.
Everyone seemed happy with their own formulation and reported good
fixation for flow. In general most stressed the need for using the fixative
soon after its preparation to reduce the possibility of formic acid
generation. From what I've read and comments in the replies, three weeks
seems to be the longest period before preparation of fresh fixative is
necessary (although at least one respondent reported good fixation with 2%
pfa kept light-protected at 4C for over a year, and another implied that
solutions were good for up to 2 yrs at 4C). The preparation buffers used
included PBS (both generic and Dulbeccos's) and Isoton. The prepared
concentrations ranged from 1% to 4% pfa, with a 1% solution being the
most common. The concentration used should probably be predicated
upon the epitope of interest as Matthias pointed out. There were differing
opinions on the pH of the final solution. Two recipes called for a pH of 7.0
while two others used a pH of 7.4. All reported good results at those pH
extremes, although one respondent pointed out that FITC is sensitive to
pHs that vary from 7.3. Temperatures for pfa solution preparation also
differed. Most heated the buffer, with stirring, to 60C though others used
70C. I'm not sure that this is a critical point. Complete solubilization of
pfa is also an issue. Paula wrote that pfa readily goes into solution in
Isoton. Others using PBS as a diluent added small amounts of either NaOH
(2 M) or KOH (5 M). Two people just used gradual heating for up to 2
hours to get most into solution and then filtered the final solution. Some
used other additives, such as d glucose, to adjust the osmolarity of the final
fixative.
By way of summary, it appears that personal preference, more than
anything, dictates the preparation of pfa fixative. The only condition that
everyone unanimously agreed on was that high quality pfa (EM grade)
should be used. The only other point that a majority agreed on was that the
final fixative solution has a relatively short shelf life (with exceptions as
noted above). There was also quite a bit of discussion on whether pfa is the
superior fixative for flow cytometry. Several people reported no
differences in quality of fixation for formalin (freshly prepared),
formaldehyde (EM grade) or pfa.
Once again, thank you for your input.
Scott
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