Sample preparation

Fixation of the tissue sample is essential to preserve tissue morphology and retain the antigenicity of the target protein during the IHC experiment and storage. Fixation can also enhance the refractive index of tissue constituents and give tissue support during sectioning.

The choice of fixative depends on the target antigen and the desired detection technique (fluorescent or chromogenic). Furthermore, the fixation method often drives the design of the sample preparation workflow; for example, tissues may need to be snap-frozen if a phosphorylated epitope is being studied. Fixation usually occurs before embedding in paraffin but after freezing.

When generating paraffin-embedded tissue samples, the tissue must be fixed before embedding in paraffin. Fixation is achieved by perfusion or immersion immediately after dissection and typically takes 4 - 24 hours. Fixation for longer than 24 hours is not recommended as it may lead to over-fixation, which may mask the antigen.

Standardized fixatives for each type of antigen are essential for reproducible staining, as an antigen that has been inappropriately fixed may not be detected. The most suitable fixative for an IHC experiment depends on the antigen, as illustrated in the figure below. Some guidelines for the type of fixative to use are given in Table 1.

After fixation, the tissue is dehydrated to enable embedding with paraffin, which is water-insoluble. The tissue is dehydrated gently by immersion in increasing concentrations of a dehydrating agent such as alcohol. This gradual change in hydrophobicity minimizes cell damage. 

Clear the dehydrating agent by incubation in xylene before paraffin embedding. Paraffin is typically heated to 60°C and then allowed to harden overnight. Finally, section the tissue using a microtome.

Tissue sections may be dried onto microscope slides and stored for extended periods at room temperature. Rehydrate the tissue sections before commencing the immunostaining protocol.

Prepare frozen samples by immersing the tissue in liquid nitrogen, isopentane, or burying it in dry ice. Snap-freezing is frequently used when detecting post-translation modifications such as phosphorylation.

After freezing, cut the tissue using a cryostat, and the resulting sections can be stored at -80°C for up to one year. Frozen tissue sections are typically fixed with an alcohol such as methanol or ethanol. As alcohols do not mask epitopes, their use avoids the need for antigen retrieval.

Formaldehyde is a gas that retains its chemical properties in an aqueous solution, whereas formalin is a saturated solution of 37-40% w/v formaldehyde in water. 10% formalin is, therefore, roughly equivalent to 4% formaldehyde.

Formalin contains ~10% methanol, which the manufacturer adds to slow the polymerization of formaldehyde in solution to paraformaldehyde. Therefore, paraformaldehyde is a solid comprised of large polymers of formaldehyde.

As the added methanol can negatively impact the fixation of certain samples, some protocols recommend making formalin from paraformaldehyde immediately before sample fixation.

Table 1. Guidelines for choosing a fixative