Hello V H Krishna Prasad

I would prefer you use formalin. If you do not have formalin, you may use glutaraldehyde. Usually, ethanol as a fixative is not recommended for this assay.

There are two basic classes of fixative:

cross-linking fixatives and

coagulant fixatives.

Formaldehyde-based fixatives are the most common type of cross-linking fixative used, and they work by covalently coupling molecules to each other and creating a stable meshwork. Glutaraldehyde is another example of cross-linking fixative.

The major disadvantage of cross-linking fixatives is that they can mask epitopes recognized by certain antibodies either by chemically modifying a critical amino acid or by blocking antibody access to the antigen because of the dense cross-linked meshwork which they generate.

On the other hand, coagulant fixatives which include ethanol, methanol, acetone or a combination are organic solvents. They function by precipitating and often denaturing proteins in situ, maintaining the structure when doing so. Organic solvent fixatives will work well when you immuno stain cytoskeletal proteins and other components of insoluble large macromolecular complexes. Organic solvents cause much greater tissue shrinkage than cross-linking fixatives and usually destroy the integrity of cell organelles. Though they initiate the fixation process much faster than formaldehyde fixatives, they destroy cell membranes thereby permeabilizing cells at the same time they fix the cells. Epitopes may be better preserved with coagulant fixatives rather than with cross-linking fixatives.

So, I recommend that you either use formalin or glutaraldehyde as fixative for colony assay.

Best.

Do you, or a colleagu's lab, have paraformaldehyde powder? You can make formalin from that. https://www.rndsystems.com/resources/protocols/protocol-making-4-formaldehyde-solution-pbs

Thank you very much for your reply Malcolm Nobre and Ellen A G Chernoff We don't have paraformaldehyde powder Ellen A G Chernoff, however, we do have glutaraldehyde. Wishing you both a wonderful year ahead