IHC Fixation Optimization

Selecting the best fixative for your sample

Fixation prevents autolysis and necrosis of excised tissues, preserving their morphology and antigenicity as well as increasing their resistance to processing. The choice of fixative is critical as different fixatives are more optimal for some antigens than others.

Antigen	Optimal Fixative
Low molecular weight peptides and enzymes, small molecules and amino acids.	4% Paraformaldehyde
Large or delicate tissues, meiotic chromosomes	Bouin’s fixative
Large proteins, nuclear/compartmentalized proteins	Acetone or methanol

Selecting the right fixation method

Once you have selected your fixative, the next step is to apply it to your sample. There are two ways to fix tissue: immersion and perfusion. Immersion is the most common method, suitable for cell cultures and excised tissue samples. The ideal amount of time for immersion fixation varies depending on the size and type of tissue being fixed: larger, denser samples will require much longer than small, light samples. Under-fixation can lead to edge staining, while over-fixation can make the epitopes difficult to unmask. Optimize your immersion time by reading papers that involve IHC with similar tissue types as yours.

While immersion fixation is usually adequate for small tissue pieces, perfusion is necessary if you must analyze larger tissues, or multiple tissue samples from the same animal. Perfusion via the vascular system can rapidly and uniformly fix all the tissues in an animal, but requires the sacrifice of the animal by replacing the animal’s blood with 4% paraformaldehyde solution.

Keywords: IHC, optimization, fixative, pfa, perfusion, immersion, fixation method

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