that you first download and read the PDF
file containing the step-by-step protocol and solution recipes.
Using that as your guide, you can then follow the protocol below and
play Quicktime movies demonstrating key steps. We have also included
higher resolution stills which provide close-ups of certain steps of
the protocol, and you can view those by clicking on their thumbnail
Feature: Complete immunostaining protocol in one Quicktime movie
to view at high resolution (17.9 Mb)
Warning: this can take a long time to load,
depending on the speed of your connection. Please be patient!
movie stars Dr. Archa Fox.
material: we grow our cells on 18 x 18 mm square or 19 mm circular coverslips
in tissue culture dishes
Rinse cells with PBS (optional step) and fix as desired. If you're testing
a new antibody, it's worth trying more than one fixation type/time.
We generally use two different types of paraformaldehyde fixation: 1.
warm 3.7% PFA in PHEM buffer for 10
min (good for preserving microtubules in mitotic cells) and 2. room
temperature 3.7% PFA in CSK buffer (5 min only for staining speckles/paraspeckles/other
splicing factors, can go to 10 min for most other antigens). We also
use a methanol fixation for certain
antibodies. Once fixed, rinse cells well with PBS.
view Quicktime movie showing the fixation and rinse steps (1.3 Mb).
Permeabilize with 1% Triton X-100 in PBS for 10 minutes at room temperature
and rinse well with PBS.
view Quicktime movie showing the permeabilization and rinse steps
Set up humidified chamber (box with a lid and water-saturated tissue in
it; bottom lined with parafilm) and place coverslips as desired (e.g.
to use low solution volumes, in the range of 20-40 ul, pipette antibody
directly onto parafilm and carefully place coverslip upside down, i.e.
with cells facing down, on top of the drop of solution; if you're using
solution volumes greater than 50 ul, you can place the coverslip on the
parafilm with the cells facing up and pipette the antibody directly onto
antibody, however, block non-specific antibody binding sites by incubating
cells for 10 min in blocking buffer (PBS + 0.1% Tween + 1% serum). After
this step, incubate cells with primary antibodies diluted in blocking
buffer for 35 mins to 1 hour. If you have never used the antibody before,
try several dilutions (low of 1:100 to high of 1:1000, for example).
view Quicktime movie showing the set-up of the chamber (1.2 Mb).
view Quicktime movie showing the two different methods of adding
antibody to the coverslips (2.9 Mb).
on thumbnail to view full-size image showing coverslips set up in
a humidified chamber
Transfer coverslips to 6-well plates for PBS washes (3 x 10 min). When
washes are done, transfer back to humidified chamber for secondary antibody
incubation). Incubate coverslips for 35 mins to 1 hour with the appropriate
secondary antibodies diluted in blocking buffer, and wash 3 X 10 minutes
with PBS. We use fluorophore-conjugated secondary antibodies from Jackson
Immunochemicals. Transfer back to the 6-well plate for final PBS washes
(3 x 10 min), and then stain with DAPI if desired (1:15,000 dilution in
water of a 5 mg/ml stock). A good RNA stain, which fluoresces in the red
channel, is Pyronin Y (1:10,000 dilution in water of a 10 mM stock; must
stain with DAPI first). Wash well with PBS.
view Quicktime movie showing the transfer of coverslips to a 6-well
plate for PBS washes (1.8 Mb).
on thumbnail to view full-size image showing coverslips in 6-well
plate for washes
Mount the cells for immunofluorescence analysis. We commonly use either
MOWIOL/Dabco, which hardens, or the liquid mounting media Vectashield
(Vector Labs), in which case the coverslips must be sealed with nail varnish).
Slides can be stored in the fridge or freezer until analyzed by microscopy.
view Quicktime movie showing the two different methods we use to mount
cells for microscopy (5.7 Mb).
on thumbnail to view full-size image showing the two different methods
we use to mount cells