Biology Reference
In-Depth Information
supplemented with 100 μg/mL gentamicin to kill the remaining extracellular
bacteria. Thereafter, incubate infected BMMs in gentamicin-free medium.
3.3. Immunofluorescence Microscopy
1. First fix infected BMMs with PFA before proceeding for immunostaining. Alter-
natively, cells can be fixed using ice-cold 100%methanol for 30 s at -20 °C when
the immunological detection of particular epitopes requires harsher extraction
conditions. The method described herein is adapted to PFA fixation.
2. Wash infected BMMs three times with room temperature PBS and then
rapidly transfer coverslips to another PBS-containing 24-well plate using high-
precision tweezers. Aspirate the PBS and immediately cover the coverslip with
300-400 μL of 2.5% PFA in PBS that has been preheated to 37ºC. Incubate
coverslips at 37ºC for 20 min (
see
Note 7
).
3. Wash fixed samples in wells three times with PBS and leave coverslips for at
least 10 min in 50 m
M
NH
4
Cl-PBS to quench free aldehyde groups. This step
diminishes the fluorescence background of the samples. Coverslips can be kept
for up to 24 h at 4 °C in quenching buffer before immunostaining although it is
preferable to perform the procedure immediately after fixation.
4. Perform all subsequent steps in a humidified, lightproof chamber such as a black
shallow box containing a sheet of parafilm on top of wet 3M Whatman paper.
5. To allow antibody access to intracellular structures and simultaneously saturate
antibody non-specific binding sites, retrieve each coverslip from its well, drain
excess liquid onto a Kimwipe, and invert it (cells facing down) on top of a
50 μL drop of permeabilization/blocking buffer. Incubate for 30 min to1hat
room temperature. Saponin is the mildest detergent for sample permeabilization
and is reversible. Hence, it is kept present in antibody dilutions subsequently
used. Alternatively, some antigens require extraction with 0.1% Triton X-100 in
PBS for 5 min at room temperature, which irreversibly permeabilizes the cells
(
see
Note 8
).
6. Prepare primary antibodies dilutions in antibody dilution buffer and distribute
them as 50-μL drops. For antibodies available in limited amounts, the volume
of the drops can be decreased to 15 μL. Drain permeabilization/blocking buffer
from each coverslip onto a Kimwipe and invert it onto the corresponding drop of
primary antibody. Gently swirl the incubation box to homogenize the antibody
drop with the remaining liquid on the coverslip. Incubate for 20 to 40 min at
room temperature depending on the antibody affinity and antigen accessibility
(
see
Note 9
and
Table 1
).
7. Prepare secondary antibodies dilutions (1:500 dilutions for all Alexa Fluor™
and Cyanin 5-conjugated antibodies) in antibody dilution buffer and distribute
them as 50-μL drops. Prepare four beakers, two containing 50-100 mL of 0.1%
saponin-PBS, one containing PBS, and one containing bi-distilled water.
8. Wash each coverslip twice in 0.1% saponin-PBS and once in PBS. Each wash
consists of holding the coverslip with tweezers and stirring for few seconds in each
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