Keep
samples (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 used).
Controls:
All experiments should include a sample that has been treated to
control for non-specific primary and secondary binding. Some controls
include:
*Incubation
with secondary antibody only (to determine non-specific staining by the
secondary antibody).
*Non-immune
serum or isotype Ig (matching the primary antibody).
*Infrequently,
some cells display autofluorescence, you may need to have a
non-processed sample to control for this.
Protocol:
1. Remove medium, follow with 3 washes of 1xPBS
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 permeabilization protocol.
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 with PBS.
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 serum).
7. Wash
monolayer 5 times with PBB.
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 with PBB.
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 with PBB
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.
Gelvatol is water soluble, if needed coverslip may be gently removed by
submerging sample in PBS and allowing coverslip to lift without
resistance from the slide. Recipe for Gelvatol on protocol website.
General recipes:
BLOCK (PBS+2% BSA) = 2g BSA per 100mL 1xPBS
PBB (PBS+0.5% BSA)= 0.5g BSA per 100ml PBS
*Other fixation protocols:
In some cases antibodies won’t work on paraformaldehyde fixed
epitopes. Other fixations include 100% cold methanol or acetone
(solvent stored at -20oC). Do not use acetone if your coverslips are
polystyrene or stored in polystyrene tissue culture dishes. Acetone
dissolves this plastic. Move coverslips to another type of
container if this type of fixation is necessary.
1. Immerse coverslips in cold
solvent for 10 min.
2. Remove and let air dry.
3. Store these at 4oC or -20oC
until processing.
4. Rehydrate cells with PBS and
wash 3 times.
5. Proceed as above for
paraformaldehyde fixed cells.
Note: Cells fixed in this manner will NOT counterstain with phalloidin.
There are many other permutations and other options available when it
comes to immunofluorescence protocols. The ones described above are
basic and work for most applications. However, if you have any
questions, or are experiencing problems, please contact a CBI staff
member for assistance. Also, may websites discuss antigen retrieval in
fixed tissues, such as: http://www.ihcworld.com/_protocols/epitope_retrieval/en_bloc.htm.
