(usually a coverslip with adherent cells) submerged in
liquid at all times until sample preparation is completed
and has been attached to a slide with Gelvatol. Store
completed samples at 4oC in the dark when not in use.
Stained samples may be rescoped for several months if stored
this way (depending on the antibodies and fluorophores
experiments should include a sample that has been treated to
control for non-specific primary and secondary binding. Some
secondary antibody only (to determine non-specific staining
by the secondary antibody).
or isotype Ig (matching the primary antibody).
some cells display autofluorescence, you may need to have a
non-processed sample to control for this.
2. Fix sample in 2%
paraformaldehyde made in PBS for 15 minutes at room
temperature. Wash monolayer 2 times in PBS to remove
residual paraformaldehyde, store samples submerged in PBS at
4oC until use. If sample(s) already have a fluorescent
label (e.g. Mitotracker, Lysotracker, Di-I-AcLDL, etc…),
then minimize exposure to light. Make sure these labels are
compatible with this particular fixation and
3. Permeabilize cells with
0.1% Triton X-100 made in PBS solution for 15 min. Different
detergent permeabilization protocols may be necessary
depending on your system. Please contact a member of
CBI with details of your system.
4. Wash monolayer 3 times
5. Wash monolayer 5 times
with a PBS+ 0.5% BSA (PBB).
6. BLOCK with 2% BSA for
45 minutes. Alternative blocks are 20% serum in PBB from the
species in which your secondary antibodies are made (e.g. if
you secondary antibody is made in goat, use 20% goat serum.
Likewise if your secondary is made in donkey, use 20% donkey
7. Wash monolayer 5 times
8. Primary antibody:
Dilute to the desired concentration in PBB (vortex gently,
spin down for 5 min at 10-12K RPM to get rid of aggregates).
Gently drop 70-100 µl antibody solution over the section (so
the section is all covered). Incubate for 60 minutes..
Longer primary antibody incubations may be necessary and
must be determined empirically in your system. Initial
antibody optimization should be accomplished in a range up
to 5ug/ml antibody in PBB. If longer incubation times
are necessary, place a piece of wet paper towel in your
slide box (or chamber) with your samples to minimize
evaporation of your primary antibody solution. In some
cases overnight incubations at 4oC are necessary, you must
put your samples in a humid environment (wet paper towel
within slide box), avoid disturbing the sample volume on
your section. Primary antibodies can be added together but
the host must be from 2 different species.
9. Wash monolayer 5 times
10. Secondary antibody for
60 minutes (made in PBB, vortex, spin down at 10-12K RPM for
5 minutes, avoid using solution from the bottom of the
vial). The secondary antibody step should be kept to
within an hour. You may add fluor-conjugated
phalloidin (for F-actin counterstain) or Draq5 (a far-red
nuclear stain from Biostatus) to your secondary solution.
Secondaries can be added together if using more than one,
but they must be against two separate species.
11. Wash monolayer 5 times
12. Hoescht stain for 30
seconds Hoechst (Sigma, cat no. B-2883) is made at
concentration of 1mg/100ml in dH2O and is stored in the dark
at 4oC when not in use. Caution! This substance is
carcinogenic and toxic to skin!!
13. Wash monolayer 3
times with PBS.
14. Adhere coverslip with
gelvatol to a slide (avoid bubbles) and store samples
horizontally at 4oC with minimal exposure to light until
use. If using coverslip-bottomed chamber slides or
Met-Tek dishes and an inverted scope, gelvatol is not
necessary. Store cells in PBS at 4oC and do not allow them
to dry out at any time until scoped.
1. Immerse coverslips in cold solvent for 10
2. Remove and let air dry.
3. Store these at 4oC or -20oC until
4. Rehydrate cells with PBS and wash 3
5. Proceed as above for paraformaldehyde