Fixation is a process that helps to lock proteins in place on cells you plan to analyze. There can be a variety of reasons to fix your cells, including:
- Timing/Covenience (experiments that cannot be completed in one day)
- Safety (biohazardous samples that must be fixed/killed prior to analysis)
- Intracellular analysis of targets (also includes permeabilization)
Fixation is a common practice, but it creates some difficulties if samples are fixed prior to staining for surface antibodies. Fixation can alter the epitope, making it difficult for your antibody to recognize its target. The information provided below is based on our in-house testing of our clones on unfixed cells (no fixation) and cells fixed with 4% paraformaldehyde prior to staining (post-fixation). We have provided data where possible. Black histograms/dot plots represent unstained samples. Purple histograms/dot plots represent staining with the indicated test antibody.
Please note that this data is based on several variables, including the types of cells analyzed, the antibody's fluorophore, and relative abundance of the antigen. Costains are also recommended to help you identify your population of interest. If you are planning to use a live/dead stain (such as Zombie Dyes), stain with them prior to fixation.
While these results will not necessarily guarantee the success or failure of staining a fixed epitope, it will at least provide you with an idea of how your samples might look.