PFA fixation and staining kit

Paraformaldehyde crosslinks are formed between protein molecules, in particular with the basic amino acid – lysine. Only those lysine residues on the exterior of the protein molecule can react with paraformaldehyde. Paraformaldehyde crosslinks are made primarily on the hydrophilic areas of proteins. The reaction between aldehyde and protein is pH-dependent, progressing more rapidly at higher pH values.

Paraformaldehyde is the condensation product of methylene glycol. It is a white solid with a formaldehyde content between 78 and 98%. Paraformaldehyde can be depolymerized to formaldehyde solution by water in the presence of a base and heat. In water, formaldehyde is slowly hydrated to form a glycol at acidic pH.

Residual formaldehyde must be quenched by primary amine such as Tris and Glycine to reduce the autofluorescence in tissue samples. Formaldehyde does not act on lipids in the cell membrane. A permeabilizing step is needed to facilitate antibody access to the cytoplasm.

Paraformaldehyde concentrations ranging from 3.7-4% are often used in fixation buffer. BiCell Scientific has found that lower paraformaldehyde concentrations such as 1-2% can better preserve antigenicity while still retaining good tissue morphology. Low paraformaldehyde content can reduce the osmotic shock to cells as well.