Fixation retains the cellular and subcellular structure by crosslinking and immobilizing of cellular proteins. The fixation leads to a stabilization of the cell morphology and e.g. the preservation of tissue architecture. Fixation of cells is mostly based on crosslinking reagents (such as Paraformaldehyde or Glutaraldehyde) typically used in concentrations of 0.1-8 %. Depending on the sample (single cells or tissues) or the structure of interest (membrane bound or located in the cytosol) the fixation conditions have to be adjusted to enable an appropriate fixation together with a sufficient staining of the respective target structure. Small amounts of Glutaraldehyde (0.05 to 0.2 %) in addition to the Paraformaldehyde/Formaldehyde strongly improves structure preservation. However, quenching of increased background fluorescence (with ammonium, NaBH4 etc.) might be necessary.

Next to crosslinking of proteins, cells can be fixed using draining reagents such as ethanol and methanol at -20 °C. However, this removes a substantial amount of lipids and membranes of the cell and leads to a loss of the cytoplasm.

Fixation of cells will immobilize antigens thus enable the labelling of cellular proteins via Immunohistochemistry (IHC). However, the strength and duration of chemical fixation has to be evaluated, because very long or strong fixation (over-fixation) can alter antigens and thus lower the staining efficiency. Depending on the antibodies themselves and the sensitivity of the epitope, the fixation method and conditions have to be optimized to enable a specific staining of the target protein. To enable a labelling of cellular proteins with antibodies, cells are normally first fixed and then permeabilized using detergents like Saponin, Trition-X or Tween-20 (which differ in their permeabilization capability) to allow antibodies to enter the cell through the permeabilized membrane.