Table 1. Parameters included in systematic evaluation of staining conditions for CD31, CD8 and FOXP3 in osteosarcoma samples.
| Retrieval | DRS pH 6, 98°C, 30 min |
| EDTA pH 7–9, 98°C, 30 min | |
| Citrat ph 6, 98°C, 30 min | |
| DRS pH 6, 110°C–136°C*, 5–15 min** | |
| EDTA pH 7–9, 110°C–136°C*, 5–15 min** | |
| Citrat pH 6, 110–136°C*, 5–15 min** | |
| Chymotrypsin, Proteinase 24, Proteinase K, Pronase, Hyaluronidase *** | |
| Blocking | No blocking |
| BSA, 5%/10% | |
| Human Serum, 5%/10% | |
| Antibodies | CD31 (JC70A) |
| FOXP3 (236A/E7) | |
| CD8 (C8/144B) | |
| Detection systems | Vectastain ABC-AP Kit (Vector Labs) |
| Vectastain Elite ABC-HRP Kit (Vector Labs) | |
| Ultravision LP detection system HRP (Polymer, Thermo Fisher) | |
| Ultravision LP detection system AP (Polymer, Thermo Fisher) | |
| EnVision+HRP labelled Polymer (Dako) | |
| Chromogens | Liquid Fast Red (Thermo Fisher) |
| Fast Red (Roche) | |
| 3,3′-Diaminobenzidine (DAB) (Vector Labs) | |
| 3,3′-Diaminobenzidine (DAB) + Nickel (Vector Labs) | |
| 3-amino-9-ethylcarbazole (AEC) (Vector Labs) | |
| Histogreen (Linaris) |
Systematic evaluation of more than 200 different antigen retrieval conditions, different blocking conditions, detection systems and chromogens allowed identification of optimal staining conditions for formalin-fixed and paraffin-embedded osteosarcoma samples.
* In steps of 3–5°C;
** in steps of 5 min;
*** in concentrations of 0.1%–10%, adjusted temperatures and various times and combinations.