Before 'puncturing' holes in the cell membrane to bring the antibodies in the cell
has to be fixated. There are two ways of fixation; cross linking (formaldehyde
and glutaraldehyde) and denaturation (acetone , ethanol and methanol). Most of the time formaldehyde is used when the cells are
going to be used in flow cytometry. This is a mild fixative that doesn't change
the morphology of the cell to much. When you are going to look at the localization
of nuclear factors on cytospins cold acetone is a perfect choice.
This denaturing agent is used most often in this kind of experiments.
Formaldehyde or glutaraldehyde.
Some proteins are more sensitive to fixation than others. We should choose
the mildest fixative that keeps the morphology of the cell and prevents the
target of leaving the cell. Most researchers use a 1 - 4% formaldehyde solution
in PBS. Others use glutaraldehyde. We prefer formaldehyde because it links
proteins less efficient, which keeps the protein structure and doesn't change
the (accessibility of the) epitopes too much. Less essential is the fixation period.
For formaldehyde this is: 10 - 15 minutes at 4°C or room temperature.
There are several stabilized forms of formaldehyde (formalin 37%) on the market.
These contain methanol and it is said that this will make the cells clot. When
the cells are fixated in a 1:10 dilution of the formaldehyde in PBS and the
cells are mixed gently on a vortex when adding the fixative and every now and than during fixation clotting
of the cells will be minimal.
Ethanol, methanol and acetone.
When you would like to look at nuclear factors or very low expressed targets
it can be useful to use a fluorescence microscope instead of a flow cytometer. It is very convenient to make cytospins which
are fixated in
-20°C acetone for 8 - 10 minutes. Fixating the cells longer than 10
minutes destroys the morphology and in the end the cells will be lost.
It is also possible to look at target in tissue sections of course. In that case
the cryo-sections are fixated in cold acetone too.