immunofluorescence of s.pombe

Michael Moser moser at U.WASHINGTON.EDU
Fri Dec 2 12:48:59 EST 1994


Dear Yeasties (and Beasties),

=09I thought the recent posting from Anne Spang definitely deserves=20
further discussion.

On 1 Dec 1994, Anne Spang wrote:

> I wondered why the protocols are so complicated and long.

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?

> why must I incubate the cells for hours (12 to O/N) with the first and th=
e
> second antibody under rotation? Isn=B4t it possible to immobilize the cel=
ls
> 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 gai=
n
> at least one day and also save antibodies

I too would really appreciate the input of anyone who has tried a version
of the above S. cerevisiae method on S. pombe.

> Why do I need to have a preculture before performing immunofluorescence?

In general freshly grown cells from a liquid culture in logarithmic phase=
=20
of growth yield better results for most microscopy experiments.

> Why do I need a  spheroblasting step with Novozym 234 and Zymolyase=20

Zymolyase alone fails to completely digest the fission yeast cell wall=20
resulting in poor staining

> AND an additional Triton 100 step?

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






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