10/11/96
mainly by immunofluorescence and am having trouble finding a method that
maintains cell morphology. Acetone gives beautiful staining, but results in
cell shrinkage. I thought I would try paraformaldehyde followed by triton
x-100. Paraformaldehyde maintains cell structure and stains surface antigens,
however, when the same cells are permeabilised by triton x-100, surface
staining is greatly reduced in intensity, cytoskeletal elements like vimentin
are weaker, tubulin does not stain at all, but von Willebrand factor is fine. I
have tried varying the time and concentration of paraformaldehyde and triton,
as well as including an extra fixation step after permeabilisation, but nothing
seems to work.
I would be very grateful for any suggestions on other permeabilisation methods.
Thanks,
Pat Taylor,
Cell Biology Group, Heart Science Centre, Harefield Hospital, Middlesex, U.K.
Pat.Taylor@HAREFIELD.NTHAMES.NHS.UK
usually helps in preservation of cell structure and surface membrane
proteins following permeabilisation by Triton X-100. In this case it may
be necessary to determine if antigenicity is also maintained after glut.
addition.
Good Luck,
Jitu
Satyajit Mayor <mayor@NCBS.TIFRBNG.RES.IN
use ice cold methanol, the second combination of PFA (3,7-4%) and them ice cold
methanol. Cells fixed in that way could be stored at -20 for several weeks.
You also can try combination PFA - detergent, but instead of Triton
X100, use more soft one (saponin for example)
Goud luck!
oleg maiboroda
oleg@MAGDEBURG.ZENIT.UNI-MAGDEBURG.DE
I did some double stainigs with kidney cells in suspension: surface
labeling and (actin or microtubules). I basically follow the protocol in
J.E. Celis' handbook (Cell Biology: a laboratory handbook, part 2, p 355).
It describes 3 fixing methods. I use the second one: 10 min 3%
paraformaldehyde, followed by 1 min 0.1% Tx-100. It gives great results
for membrane staining as well as the cytoskeletal parts. The EGTA is of
major importance for the tubules.
greetings
herman favoreel
lab virology
university of gent
belgium
Herman.Favoreel@RUG.AC.BE
We routinely fix our endothelial cell cultures with 3.7%
paraformaldehdyde in PBS and then permeabilize with digitonin (approx.
60 fg/ml) before blocking and then treating with primary antibody. We
use this protocol to stain for a number antigens including vimentin,
tubulin, and beta 1 integrin subunit. If you would like a more
detailed protocol please e-mail me directly.
Bryon Grove
Bryon Grove,
Dept. of Cellular Biology and Anatomy,
L.S.U. Medical Center,
1501 Kings Hwy,
Shreveport, LA, 71130
e-mail: bgrove@nomvs.lsumc.edu