3/22/97
species of sulfate-reducing bacteria using ConA-conjugated gold (5nm).
Using 4%PF+0.1%Glute in Cac buffer fixation followed by LRW embedding I
have tried 1:10 to 1:100 in P04 buffer pH 7.2. I have also tried ConA-gold
in Tris-saline with and without 1mM CaCl2 and 1mM MgCl2 at pH 6.9,7.2
and 8.0. diluted 1:10-1:100. Next I tried taking a fresh culture and
washing several times in Tris-saline followed by 1:10 ConA-gold,then
fixing and embedding. My incubations have been around 1 hr at room temp.
Nary a gold particle do Isee under any of the conditions mentioned.
Apparently, using fluorescently labelled ConA these little buggers lightup
like the Hale-Bopp comet, so Iam at a lost. Can anybody out there give me
some suggestions. I also have some material that I fixed in 4% PF alone,
embedded in LRW that I have not done anything yet with.
Thanks in advance
Hank Adams
Electron Microscopy Laboratory
New Mexico State University
Las Cruces, NM 88003
hadams@nmsu.edu
Your problem may lie in the ConA-conjugated gold. Often, these probes very
seldom store well. If most of the protein has dissociated from the gold
particles, you will have a large amount of very specific ConA binding but no
gold to show you it is there. It is easy to test at the LM level by probing
sections treated with ConA-god with anti-ConA and fluorescent antibody.
Once again, the best visualization method for lectins (re:ricin) may be to use
antibodies instead of the directly conjugated gold probes. If you prefer to use
ConA-gold then I recommend that you make your own. A slightly less satisfactory
approach would be for you to centrifuge down your ConA-gold (be careful not to
spin it so hard that is aggregates into the bottom of the tube) and resuspend it
in fresh PBS. This will remove most of the free protein.
As an aside and in reference to a previous posting, streptavidin-gold also
suffers from this storage problem. When using the probe at the LM level, with
silver enhancement, or with multiple antibody layers the loss of labeling
efficiency is not so noticeable. However, attempting to visualize biotinylated
antibodies directly with streptavidin-gold will only work well for the first
week after the probe has been made. For situations where there are large
numbers of bound antibody, this effect may not be noticeable, but for situations
where there are only low numbers of antigens, it is very obvious.
Making colloidal gold and coupling it to proteins or antibodies is easy and
anyone with even limited experience can do it.
Check out my WWW "gold page" to see how easily it can be done. Be warned, if
you want to do this you usually need large supplies of protein (a point anyone
coupling antibodies should be aware of).
Paul Webster
Center for Cell Imaging
Yale School of Medicine
paul.webster@Yale.edu
of sodium periodate which will destroy the section if you are not
careful.. This will expose some of the bnding sites fo the Con-A.. or
on the other hand the Lctins are too old, and the gold has fallen off
of the lectin which occured to me also when I was using gold labeled
lectins that were stored in the fridge........ So I got very
scattered labeling or no labelling at all but when used in teh LM
with Cy3 they light up like a chrsitmas tree....
lAlso you may want to try a lower comcentration of fixatives in
Formaldehyde and Glut.
Eric
earosen@pop.goodnet.com
to worry about sectioning to expose the conA to the glycocalyx, why don't
you stain with con A-Au, wash thoroughly (centrifugations), and then embed
normally?
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
A, keep the pH high (probably above 8-8.2, or whatever pH the original
stuff was shipped in). Otherwise, you might have one big blob at the
bottom of your tube. When we suspect unconjugated protein, we dilute the
antibody-Au to the concentration we want to use with buffer at a pH above
the pI (pH > 8), and then spin in a Beckman Airfuge 5 min at 30 lb
(~100,000 g), discard the sup and resuspend the pellet in the same vol(pH 7)
as we started with. Haven't tried con A; I assume it might work the same
way???
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 you want to test your fixation, try various ones and then fluorescent
labeling--saves a lot of work doing it at the EM level.
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
are usually not permeable to gold labels (ie you only present a very small
edge of the bacteria in e.m. whereas I assume the light microscopy that you
mention was done on whole cells which present a much larger surface to the
fluorescent label. One test might be to try and use the fluorescent label on
LR White sections and examine that under the fluorescent light microscope.
Malcolm Haswell
University of Sunderland
UK
es0mhs@environment.sunderland.ac.uk