After proper fixation of tissue, there are some important tissue processing and microtomy principles that must be followed to assure quality IHC staining.
- Heat applied during processing should not exceed 60° C to preserve antigenicity, as proteins can be altered by excessive heat.
- During microtomy, additives in the water bath should be avoided and deionized water should be used in the bath to avoid any non-specific staining later.
- Tissue sections should be cut at 3-4 microns and tissue should be free of wrinkles, folds, and air bubbles under sections. Thickness in tissues could pick up additional stain and make interpretation difficult. Wrinkles, folds, and air pockets can trap additional reagents that cause unnecessary artifact on the slides.
- A positively charged slide is preferred for tissue sections to help ensure adhesion, especially if a pretreatment, such as antigen retrieval, is required. Pretreatment can be harsh and cause washing of the tissue if not on a charged slide.
- Once tissue sections are placed on the slide, the slides should be placed in an oven for 30 minutes at no higher than 60° C to dry the excess water. Excess water or trapped water under tissue sections can cause washing during staining. It is important to monitor this temperature as excessive and high heat may cause weak or false-negative staining, particularly for nuclear and cell surface markers that are especially sensitive.