In addition to ensuring adequate fixation of tissue samples, there are some important tissue processing and microtomy principles that must also be followed to assure quality IHC staining.
- To preserve antigenicity, heat applied during processing should not exceed 60° C as proteins can be altered by excessive heat.
- During microtomy, deionized water should be used in the bath to avoid any non-specific staining later. Additionally, additives in the water bath should also be avoided for the same reason.
- Tissue sections should be cut at 3-4 microns. Tissue should be free of wrinkles, folds, and air bubbles under sections. Tissue sections that are too thick can pick up additional staining reagents and make interpretation difficult. Wrinkles, folds, and air pockets can trap additional reagents that cause unnecessary artifacts on the slides, as well.
- 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 section, thus the need for greater adhesion.
- 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 the tissue section to wash off during staining. It is important to monitor oven temperatures as excessively high heat may cause weak or false-negative staining. This is particularly true for nuclear and cell surface markers which are especially sensitive to heat.