Fixation is performed to keep the cell/tissue sample to be in its original form and characteristics, so that during the experiment the sample can stay in a permanent condition. Fixation agents works in different ways, for example covalent cross-links between molecules can be initiated by Paraformaldehyde, which effectively attached them together to form an insoluble meshwork. For immunostaining, the staining reagent need to permeabilize into the cells. But if the cells are not fixed, the structure of the cell shape may change which may cause the staining reagent to be diffused away before the incubation has been finished. Also, the sample should be in small volume to allow better access of the fixation solution and the fixation solution volume should be at least twenty times more than the specimen volume. After fixation, the sample should be washed to remove the fixation traces completely unless it may hinder proper staining of the sample. Before fixation, please check the cell type and size to decide the type and volume of fixation solution. Regards.