10/3/96
I would appreciate some pointers from anyone who is familiar with
fixation of cultured cells to be subsequently used immunogold
labeling. I have tried using 4% paraformaldehyde, 0.1%
glutaraldehyde in PBS @ pH7.4 but the cell morphology was quite
chewed up. Thanks for your help.
Philomena Kaan
UBC Pulmonary Research Laboratory
St. Paul's Hospital
Vancouver, B.C.
PKaan@prl.pulmonary.ubc.ca
I suggest that you increase the concentration of glutaraldehyde to 1%. (4%
PFA + 1% GA).
When I fix cultured cells, I heat the fixative to 37 degr. C, and fix the
cells taken directly out of the chamber -after one wash with buffer of the
same temperature -(if they're not free-floating). I let them cool down in RT
for a while, and then put them in the fridge for a couple of hours / over
night. On the other hand; when you embed the cells in plastic that is
suitable for immuno-gold staining + omit the osmium treatment, you don't
really see what you're used to...
Good luck!
Margareta.Halin@ah.slu.se
Dept. of Anatomy and Histology
Sveriges LantbruksUniversitet
2% freshly depolymerized paraformaldehyde (NOT formalin!)
70 mM NaCl
30 mM HEPES
5 mM CaCl2
pH 7.4
Cells like divalents (Ca2+) around during fixation.
The osmolarity needs to account for the initial contribution of the
aldehydes (a tricky situation since they are somewhat permeable but this
increases with time.
The infiltration into plastic should be gradual (2:1 and 1:1 solvent:resin).
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
antigenic sites. The general rule is that the higher the glutaraldehyde
concentration, the better the ultrastructure, but the worse the
immunostaining. The only way to know if your particular antigen remains
active after glut is to try it. Usual percents used range from about 0.1
to 1%, mixed with paraformaldehyde (4-8 %). Also, sometimes staining
decreases with increased storage of specimens in aldehydes, even
formaldehyde. If you're starting with an unknown, your best bet is to
keep the glut low (we don't use it), and the fixation time short (1-2
hrs). Then you can test to see if more glut, longer fixation times, or even
osmium fixation (not usually used except in freeze substitution), can be
tolerated. Margareta's statement that the ultrastructure of
non-osmicated tissue is not what you're used to seeing is very true.
Maybe your cells aren't chewed up--just not stained conventionally.
Sara E. Miller, Ph. D.
P. O. Box 3020
Duke University Medical Center
Durham, NC 27710
Ph: 919 684-3452
FAX: 919 684-8735
saram@acpub.duke.edu
If the cells are to be post embed labelled I would worry about too strong
of fixation in fear of loosing the immunolabelling capacity.
You might try a 2-4% para in sorensons (so there is no NaCl in the
formula) or a combination of para and picric acid in sorensons buffer such
as a Zamboni fixative, if you are post embed labelling on sections.
We have noticed a problem when there is NaCl present in the fix, with poor
morphology.
Bob
Morphology Core
Derm
U of Wash
Seattle
underwoo@u.washington.edu
results fixing in PBS. We generally use 3% freshly (that's important)
depolymerized formaldehyde and 0.05% glut. in a modified Hanks buffer.
The more glut. your antigens will tolerate, the better. To get the
membrane contrast closer to what you are used to seeing by
conventional EM,try en bloc staining with Uranyl Acetate (which can help
to improve the retention of antigenicity) and Tannic Acid (which can
reduce antigenicity, so try some tissue +TA and some -TA). The
composition of the modified Hanks fixative and protocols for en bloc
staining can be found in:
J. Histochem. Cytochem. 40:845-857 '92.
There is (are?) a myriad of protocols for immunoEM. I sort of
inherited this one and found it to work very well for many cell types and
antigens, using either LRGold or Lowicryl K4M resins.
Happy Labeling!
Greg Martin
gmartin@welchlink.welch.jhu.edu
Dept. Cell Biology and Anatomy
Johns Hopkins School of Medicine
Here's a tip that will help your unosmicated/immunostained sections look
more aesthetically pleasing. After you have immunostained your
sections, presumably they are on nickel or gold grids, run the grids
through a series of droplets which parallel a routine fixation: hard fix
in 1-2% GA, buffer washes, OsO4, washes, UA, wash, blot dry and scope.
You need only incubate for 10-15 min. in the fixes and 5 min. in the
washes and so on. I've done this with LR White and Lowicryl and it
works well. My colleagues see fine structure which more closely
resembles "routinely" fixed TEM specimens and have their imunno-gold
staining as well. Kind of like having your pie and eating it too. Try
it - you'll like it!
Cheers,
John Aghajanian
Worcester Foundation for Biomedical Research
JOHNA@SCI.WFBR.EDU