Fixation

Cell source Stimulation Secretion stop Fixation Permeabilisation Staining Protocol

 

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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 4C 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 -20C 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.

 

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Page was last updated: 05-03-2005