Immunofluorescence of S . cerevisiae
50 ml of an exponentially growing yeast-culture and spin the cells down for 5'
at 10000 rpm in the Sorvall centrifuge. Take the pellet up in 20 ml.
very slowly 2 ml 38%
formaldehyde (final concentration is 3.5%). Leave the cells for 2 to 4 hours at
the cells 5 times in TBS, for example in 20 ml.
the cells in 500 Ál zymolyase buffer and put the suspension in an Eppendorf
tube. Add 5 Ál Β-mercaptoethanol
and 10 Ál 5 mg/ml zymolyase to break down the cell wall partially.
the cells at 30íC and keep checking the morphology of the cells with a phase
contrast microscope. After 10-30 minutes the cells are looking grey, than they
are ok. If the incubation is too short, the antibody won't get in and if the
incubation is too long, the cells will become so fragile that they will degrade
during spinning down and resuspending, so that you end up with very few cells
that are easily crushed under the microscopic slide. From now on all
the centrifuge work must done in the Eppendorf centrifuge at 7000 rpm.
the cells 4 times in 500 Ál TBS
the cells in 500 Ál TBS and add 50 Ál 38% formaldehyde. Leave for 30 minutes at
three times in TBS, resuspend in 500 Ál acetone and incubate for 5 minutes at
-20íC. Not longer than 5 minutes, acetone makes holes in the membranes!
Alternatively, if you think acetone treatment is too drastic, it can be
replaced by incubating with Triton X100.
- Wash three
times with TBS, resuspend in 500 ml TBS containing 0.5% (final) Triton X100 at
room temperature for 15 min.
the cells 3 times in 1.2 M sorbitol and resuspend in 500 Ál. In this state, the
cells can be stored in the freezer for several weeks. In case the cells do not
need to be stored, the cells do not have to be resuspended in sorbitol but can
just be washed twice in TBS.
50 Ál of the cells (or more if there are few cells) and wash twice in TBS.
Resuspend in 50 Ál.
the first antibody, the dilution depends on the antibody. Incubate for one to
two hours at 37 °C.
three times in 200 Ál TBS, resuspend in 50 Ál.
the second antibody (coupled to fluorescent dye). Usually the dilution is 1:50,
so add 1 Ál. Incubate 1 hour in the dark to prevent bleaching.
3 times in TBS to get rid of non-bound antibody and resuspend in a small volume
to get a reasonable amount of cell on your slide.
3 Ál on a microscopic slide, spread it out and let dry. This prevents the cells
from floating. Add a drop of antifade to the dryed cells, put a coverslip on it
and press slightly on it with a dot of tissue, to absorb the excess of
antifade.Prevent the slide from drying out with nail polish. Examine with a
fluorescence microscope (A more gentle way of preventing the cells from
floating is to mount the cells on a thin layer of 1%agarose or mixing them with
an equal volume of 2% LMP agarose (ask Jos), but then the cells must already
have been resuspended in antifade). Slides treated with nailpolish can be
stored at -20 up to several weeks without considerable loss of quality.
Antifade: dissolve 100mg p-phenylenediamine (very
poisonous!) in 10ml TBS, add 1ml of this to 9ml glycerol, mix well, eventually
add 20ng/ml DAPI for simultanious DNA-staining. Antifade should be stored at
-20 °C, prevented from light.
- TBS : Tris buffered saline: 10 mM tris, 150 mM NaCl,
- Formaldehyde 38%
- Zymolyase buffer: 1.2 M sorbitol, 0.1 M tris, pH
- Zymolyase 5mg/ml
Remarks - abbrevations
|Always be carefull with removing the supernatant.
Because of the many washing-steps you will loose a lot of cells! Add a drop TY
as wetting agent!|
|Instead of TBS, in my opinion, PBS can also be used|