TABLE 1.
IHC Protocol for formalin fixed paraffin embedded IVD tissue all suppliers are provided in Table 2
| Step | Action | Timings |
|---|---|---|
| 1 | De‐wax sections in sub‐X | 3 × 5 min |
| 2 | Rehydrate sections in 100% IMS/ethanol | 3 × 5 min |
| 3 |
Block endogenous peroxidases: Submerge in 100% IMS/ethanol containing 3% (v/v) H2O2 and 0.06% (v/v) of concentrated HCl |
30 min |
| 4 | dH20 | 5 min |
| 5 | TBS (20 mM Tris, 150 mM NaCl, pH 7.5) wash (on flat bed shaker) | 3 × 5 min |
| 6 | Antigen retrieval: | |
| None: proceed to step 8 | ‐ | |
| Enzyme: Preheat 1× TBS buffer containing 0.1% (w/v) CaCl2 dihydrate and 0.01% (w/v) α‐chymotrypsin at 37°C | 30 min | |
| Heat: Preheat buffer containing 0.05M Tris, pH 9.5 to 60°C. Submerge slides, irradiate for 5 min at 40% power in 800 W microwave, leave to stand for 1 min, irradiate at 20% for another 5 min. Leave to cool for 15 min at room temperature. | 26 min | |
| 7 | TBS wash (on flat bed shaker) | 3 × 5 min |
| 8 | Block nonspecific antibody‐protein interactions | 1–2 hours |
| 9 | Remove excess blocking solution by tapping slides on tissue paper. | ‐ |
| 10 | Apply primary antibody or appropriate IgG controls | Overnight 4°C |
| Day 2 | ||
| 11 | TBS wash (on flat bed shaker) | 3 × 5 min |
| 12 | Apply conjugated secondary antibody (Table 3) | 30 min |
| 13 | TBS wash (on flat bed shaker) | 3 × 5 min |
| 14 | Apply ABC elite reagent | 30 min |
| 15 | TBS wash (on flat bed shaker) | 3 × 5 min |
| 16 | Apply DAB solution | 20 min |
| 17 | H2O wash (running cold tap water) | 5 min |
| 18 | Counterstain nuclei with Mayer's hematoxylin | 1 min |
| 19 | “Blue” sections under running cold tap water | 5 min |
| 20 | Dehydrate sections in 100% IMS/ethanol | 3 × 5 min |
| 21 | Clear sections in sub‐X | 3 × 5 min |
| 22 | Mount sections with coverslips using a mounting medium compatible with sub‐X, for example, Pertex or DPX | ‐ |