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With the cells plated on the previous day and cultured overnight, we are ready
to start with the ICC procedure.
On this first day of ICC, we will fix,
permeabilize,
blot and add a primary antibody to the cells.
Start by aspirating culture medium from each well followed by fixation of the
cells with 4% formaldehyde or 10% formalin
for 10 minutes at room temperature.
Aspirate fixative and rinse each well twice with ice cold PBS.
Be careful not to let the cells dry out in this
or any further step of the protocol.
If the epitope of your protein of interest is expressed intracellularly,
cellular permeabilization is necessary for the antibody to gain access to the
cell.
Although there are many different permeabilization agents, the most common
are .1-.5% concentration of Tween-20 or
Triton-X100
diluted in PBS.
Tween-20 is used for epitopes located in cytoplasm
while Triton-X100 is used for permeabilizing the nucleus and mitochondria.
Incubate wells with permeabilization buffer for 10 minutes at room temperature.
Aspirate permeabilization buffer and wash three times for 5 minutes
each with PBS twine.
PBS twine contains .1% Tween-20.
We will now block unspecific binding sites with blocking buffer for 1 hour
at room temperature.
The best blocking buffer is PBST containing 10% serum from the
host species of your secondary antibody.
However, 1% BSA in PBST may also be used.
Prepare the primary antibody by diluting it in blocking buffer at the recommended
dilution specified on the datasheet.
Multiple tighter dilutions are beneficial to account for variables that
may be different in your assay and
for achieving the best results.
Incubate at 4 degrees Celsius overnight.
In our example, since we are using an unconjugated primary we will be using a fluorophore
conjugated secondary on day two.