11/13/97
We want to change our fixation and processing protocol for pulmonary tissues
and cells to use HEPES buffer instead of sodium cacodylate buffer. Is anybody
out there who has made her/his experience with HEPES buffer in conventional
epoxy resin embedment. We are especially interested in experience of lipid
retention/extraction.
Heinz Fehrenbach (Inst. of Pathology, TU Dresden, Germany)
hefeh@Rcs1.urz.tu-dresden.de
Paraformaldehyde/Glutaraldehyde buffer and for my osmium buffer. Safer,
better for environment, and cheaper (I think). I noticed no difference
compared to cacodylate (which I had previously used). A couple of years
ago, I got into a discussion in which a colleague was touting PIPES as a
better alternative. The logic is that HEPES has a pKa of 7.5. I use it at
7.4 for my fixes. Aldehyde fixes tend to acidify over time, therefore,
since you start on the low side of the pKa and continue to fight a drop on
that side, you have less buffering capacity. PIPES on the other hand, has
a pKa of 6.8 so if you started at 7.4, you would have more buffering
capacity (0.5 on both sides of the pKa). This is good logic but I didn't
bother switching. Good luck.
Thomas E. Phillips, Ph.D.
Associate Professor of Biological Sciences
Director, Molecular Cytology Core Facility
3 Tucker Hall
University of Missouri
Columbia, MO 65211
(573)-882-4712 (voice)
(573)-882-0123 (fax)
tphillips@biosci.mbp.missouri.edu
for NaCacodylate for TEM during the past 6 months. So far the results have
been good for mammalian and amphibian tissues (cardiac muscle, pancreas,
heart, tongue). The membranes look good and we seem to have no trouble
retaining lipid droplets in frog atrial muscle. I would be interested in
anything anyone else has to offer. Also, I am assuming that HEPES is less
toxic than Cacodylate. If anyone knows otherwise, I would like to hear
about it.
Dr. Marie E. Cantino
Dept. of Physiology and Neurobiology, U-131
University of Connecticut
Storrs, CT 06269
Ph: 860-486-3588
Fax: 860-486-1936