immunofluorescence of s.pombe

Tom Chappell dmcbtch at ucl.ac.uk
Tue Dec 6 13:03:05 EST 1994


In article <spang-0112942147420001 at genmac4.biochem.mpg.de>
spang at vms.biochem.mpg.de (Anne Spang) writes:

> Hi yeasties!
> 
> Could somebody out there send me a protocol for immunofluorescence of
> fission yeast s.pombe or at least answer my questions?
> I have course manual book "experiments with fission yeast" from Alfa,
> Fantes Hyams, McLeod and Warbrick. Of course, there are 2 protocols
> included. But since I am usually performing immunofluorescence with
> S.cerevisiae, I wondered why the protocols are so complicated and long. So
> why must I incubate the cells for hours (12 to O/N) with the first and the
> second antibody under rotation?Isn´t it possible to immobilize the cells
> on a polylysine-coated slide, incubate for 2 h with the first and also
> with the second antibody? Only with this simple modification, I would gain
> at least one day and also save antibodies. Why do I need to have a
> preculture before performing immunofluorescence? Why do I need a
> spheroblasting step with Novozym 234 and Zymolyase AND an additional
> Triton 100 step?
> 
> Thanx for help!
> Anne


In article
<Pine.A32.3.91b.941202092736.24676A-100000 at homer06.u.washington.edu>
moser at U.WASHINGTON.EDU (Michael Moser) writes:

> I've wondered why they are so long as well.  Anybody out there know more=20
> about these procedures?  For example why use sodium borohydride 3X to=20
> reduce the glutaraldehyde following fixation?
> 
> I too would really appreciate the input of anyone who has tried a version
> of the above S. cerevisiae method on S. pombe.
> 
> 
> Zymolyase alone fails to completely digest the fission yeast cell wall=20
> resulting in poor staining
> 
> 
> S. pombe may have a different membrane lipid composition than S.=20
> cerevisiae (anybody correct me if this is incorrect).  The Triton wash is
> supposed to remove any lipid remaining in the fixed cell preparation
> following cell wall hydrolysis.  I have found that elimination of the
> Triton wash can result in poor DAPI staining of S.  pombe cells mounted on
> polylysine coated slides.
> 
> Thanks again for any other good ideas and suggestions people can offer on=
> =20
> the subject of new and improved S. pombe immunofluorescence techniques
> 
> 
> Mike Moser                                             Tel: 206-543-5354
> Department of Biochemistry  SJ-70                      FAX: 206-685-1792
> University of Washington                          moser at u.washington.edu
> Seattle, WA  98195                          Make peace my beast is yeast
> 
> 
> 

Having done some S. pombe immunofluorescence (and taught it for the
last 3 years at the Cold Spring Harbor course) I'd like to add a few
comments to Anne and Mike's questions.

Staining protocols are very dependent on the specific antibody used.
The O/N incubations with the YOL1/34 rat monoclonal for tubulin
staining is important to get good staining. This is probably due to 2
factors. First, the cells are fixed for a relatively long time with
both form. and glut. to get good retention of tubulin structures. This
fixation gives a very "tight" matrix of crosslinked proteins that the
YOL1/34 antibody only slowly penetrates. Second, the spindle pole is in
the center of this tight matrix of protein. Incubation at 37 C may help
to speed up the YOL1/34 staining, I haven't tried it.

Some of the protocols in the CSH manual are designed more for the
timing of the course than how one would actually do the labeling on a
day to day basis. In general, methanol fixation and form. only fixation
should give faster antibody penetration than form./glut. fixation.

With Golgi specific antibodies and form. only fixation, I typically do
a 2-4 hour incubation in primary antibody and a 1-2 hour incubation in
secondary antibody. I usually do the labeling with a 1 microliter
pellet of cells (about 5,000,000 cells) in a total volume of 10-50
microliters. 

Removal of the cell wall is important in order for antibodies to
actually penetrate into the cell. With a glut./form. fixation, you can
get away with removing the cell walls almost to completion without
destroying the fixed cells. With form. only or methanol fixation,
overdigestion of the cell wall will start to chew up the fixed cells.
This is probably due to the presence of all kinds of proteases and
other nasties in the Novozym 234. Pure Zymolyase 100T doesn't do an
effective job of cell wall digestion. The CSH manual suggests
monitoring the wall digestion by the change in phase contrast. I find
this difficult and much prefer to watch the digestion progress using
100x DIC. You don't actually digest the cell wall, but rather digest
away at the fission scars and the wall then slides off of the cell.
With 100x DIC you can see directly the walls sliding off the cells and
the difference between cells with and without walls. Given the somewhat
phallic shape of pombe cells, the best analogy I can draw to the
removal of the wall involves condoms, as does everyone who watches the
walls slide off while I'm trying to teach this...

After cell wall removal, you have to remove the lipids of the plasma
membrane, ER, nucleus, etc. in order for antibodies to get into the
cells. This is what the TX-100 wash is for. You shouldn't have removed
lipid during glut./form. or form. only fixation. Methanol fixation will
remove all the lipid in the initial fixation step (making the cell wall
digestion all the more critical--with methanol fixation you allow the
Novozym and Zymolyase into the cells...)

DAPI should get into the nucleus of any cells (fixed/unfixed; with or
without cell walls; TX-100 washed or not).

You can get away with binding the cells onto slides after the TX-100
step. It doesn't appear to be as effective as binding S. cerevisiae to
slides, I have a protocol from Robin Wright using 1% PEI for binding S.
cerevisiae to slides that she says is very effective (nearly all cells
stay on the slide during washes). In my hands with S. pombe, the PEI is
better than poly-L-lysine, but most of the cells end up washed away at
the end of the protocol. You have to be very careful in doing the
washes. You cannot dip the slides in the wash solutions and have any
cells left on the slide at the end... If anyone has found something
better, I'd love to hear about it. I've thought of binding the cells
down on lectins, but haven't tried...

Tom Chappell
MRC Laboratory for Molecular Cell Biology
University College London 

 
















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