That is what it is all about; STAINING. Showing the target is there and
with a bit of luck you can pin point the place the target is in (using fluorescence
microscopy). The staining is depending on
all the other factors; cells, inhibition of secretion, fixation,
permeabilization and the antibodies.
The staining can be performed at room temperature. Since the cells are
stabilized by the fixative. And the diffusion is higher with a higher
temperature. This way antibodies can go more easily in and out of the cells.
Some fluorochromes are temperature sensitive though, like the PerCP conjugates.
Should you use this kind of fluorochromes incubation on a bed of ice or in the
fridge is the solution. Incubate in all cases in the dark. That way you
fluorochromes last longer.
And when you think you thought of
everything something went wrong......... We are still working on the 'chapter' of