Table 1.
Immunohistochemistry Protocols.
| Antigen | Antigen Retrieval | Primary Antibody | Blocking | Visualization System | Chromogen | Washing Buffer |
|---|---|---|---|---|---|---|
| CD34 | Target Retrieval Solution (S1699, Dako); 20 min at 80°C | Goat polyclonal CD34 antibody (sc-7045, LOT H2214, Santa Cruz Biotechnology) 1:50; overnight at 4°C | 5% dried skim milk in PBS, pH 7.4; 1 hr at room temperature | LSAB+ Kit (K0679; Dako) | 3-amino-9-ethylcarbazole (AEC+); 5 min at room temperature | TBS-T |
| Sox9 | Tris-EDTA buffer, pH 9.0; 15 min in a pressure cooker | Rabbit polyclonal Sox9 antibody (sc-20095, LOT I1213, Santa Cruz Biotechnology) | 5% dried skim milk in PBS; 1 hr at room temperature | EnVision+ Kit (K4008; Dako) | AEC+; 10 min at room temperature | TBS-T |
| 1:150; 2 hr at room temperature | ||||||
| Keratin15 | None | Mouse monoclonal Keratin 15 antibody (MS-1068-PABX, LOT 1068X1006A, Thermo Scientific) | 4% bovine serum albumin in PBS; 1 hr at room temperature | EnVision+ Kit (K4004, Dako) | AEC+; 5 min at room temperature | PBS-T |
| 1:1500; 1 hr at room temperature | ||||||
| LGR5 | Sodium Citrate buffer pH 6.0; 20 min at 80°C | Rabbit polyclonal LGR5 antibody (HPA012530, LOT B83270, Sigma-Aldrich); | 5% dried skim milk in PBS; 1 hr at room temperature | EnVision+ Kit (K4008, Dako) | AEC+; 10 min at room temperature | PBS-T |
| 1:1200; 1 hr at room temperature | ||||||
| Nestin | Sodium Citrate Buffer pH 6.0; 25 min at 90°C; 15 min in absolute methanol | Rabbit polyclonal Nestin antibody (N1602, LOT 1C-307, IBL Int.); | 5% normal goat serum and 3% dried skim milk in PBS-T; 1 hr at room temperature | EnVision+ Kit (K4008, Dako) | AEC+; 10min at room temperature | PBS-T |
| 1:200; overnight at 4°C |
Abbreviations: TBS-T, Tris-buffered saline with 0.05% Tween-20, pH 7.5; PBS, phosphate-buffered saline, pH 7.4; PBS-T, phosphate-buffered saline with 0.05% Tween-20, pH 7.4.