| Lymphoid panel: | ||||
|---|---|---|---|---|
| Cycle 1 | Cycle 2 | Cycle 3 | Cycle 4 | |
| Round 1 | Round 1 | Round 1 | Round 1 | |
| Target | CC3 | PD-1 | TCF1/TCF7 | CD3 |
| Company/Product | CST (9661S) | CST (84651) | CST (2203S) | Thermo Fisher (RM-9107-S) |
| Clone | polyclonal | D7D5W | C63D9 | SP7 |
| Dilution | 1/100 | 1/100 | 1/200. | 1/300 |
| Duration | RT, 1 hr | ON, 4°C | RT, 1 hr | RT, 1 hr |
| Secondary species | Rabbit | Rabbit | Rabbit | Rabbit |
| AEC | 1 h 5 min | 1 h 20 min | 15 min | 53 min |
| Round 2 | Round 2 | Round 2 | ||
|---|---|---|---|---|
| Target | Hematoxylin | CD45 | CD8 | |
| Company/Product | Dako (S330130-2) | BD Bio (550539) | eBioscience (14-0808-82) | |
| Clone | NA | 30-F11 | 4SM15 | |
| Dilution | ready to use | 1/50. | 1/100 | |
| Duration | 1 min | RT, 1 hr | ON, 4°C | |
| Secondary species | NA | Rat | Rat | |
| AEC | NA | 52 min | 30 min |
| Cycle 5 | Cycle 6 | Cycle 7 | Cycle 8 | |
|---|---|---|---|---|
| Round 1 | Round 1 | Round 1 | Round 1 | |
| Target | PD-L1 | CD11b | Granzyme B | Ki-67 |
| Company/Product | CST (13684) | Abcam (133357) | Abcam (4059) | Abcam (15580) |
| Clone | E1L3N | EPR1334 | Polyclonal | Poylclonal |
| Dilution | 1/50 | 1/3,000 | 1/200 | 1/1,000 |
| Duration | ON, 4°C | RT, 1 hr | ON | RT, 1 hr |
| Secondary species | Rabbit | Rabbit | Rabbit | Rabbit |
| AEC | 23 min | 17 min | 10 min | 24 min |
| Round 2 | ||||
|---|---|---|---|---|
| Target | panCK | |||
| Company/Product | Abcam (ab27988) | |||
| Clone | AE1/1E3 | |||
| Dilution | 1/100 | |||
| Duration | RT, 1 hr | |||
| Secondary species | Mouse | |||
| AEC | 22 min |
| Round 3 | ||||
|---|---|---|---|---|
| Target | Hematoxylin | |||
| Company/Product | Dako (S330130-2) | |||
| Clone | NA | |||
| Dilution | Ready to use | |||
| Duration | 1 min | |||
| Secondary species | NA | |||
| AEC | NA |
| Myeloid panel: | ||||
|---|---|---|---|---|
| Cycle 1 | Cycle 2 | Cycle 3 | Cycle 4 | |
| Round 1 | Round 1 | Round 1 | Round 1 | |
| Target | F4/80 | CSF1R | CD11c | CD206 |
| Company/Product | Serotec | Santa Cruz | CST (97585) | Abcam (64693) |
| Clone | CI:A3-1 | E2412 | D1V9Y | polyclonal |
| Dilution | 1/200 | 1/1,000 | 1/100 | 1/2,000 |
| Duration | RT, 1 hr | RT, 1 hr | RT, 1 hr | RT, 1 hr |
| Secondary species | Rat | Rabbit | Rabbit | Rabbit |
| AEC | 22 min | 16 min | 15 min | 43 min |
| Round 2 | Round 2 | |||
|---|---|---|---|---|
| Target | CD45 | MHCII | ||
| Company/Product | BD Bio (550539) | eBioscience (eBi14-532) | ||
| Clone | 30-F11 | M5/144.15.2 | ||
| Dilution | 1/100 | 1/200 | ||
| Duration | ON, 4°C | ON, 4°C | ||
| Secondary species | Rat | Rat | ||
| AEC | 30 min | 63 min |
| Cycle 5 | Cycle 6 | Cycle 7 | ||
|---|---|---|---|---|
| Round 1 | Round 1 | Round 1 | ||
| Target | PD-L1 | Ki-67 | CD11b | |
| Company/Product | CST (13684) | Abcam (15580) | Abcam (133357) | |
| Clone | E1L3N | Polyclonal | EPR1334 | |
| Dilution | 1/50 | 1/1,000 | 1/6,000 | |
| Duration | RT, 1 hr | RT, 1 hr | RT, 1 hr | |
| Secondary species | Rabbit | Rabbit | Rabbit | |
| AEC | 23 min | 24 min | 8 min |
| Round 2 | Round 2 | Round 2 | ||
|---|---|---|---|---|
| Target | Ly6G | panCK | Hematoxylin | |
| Company/Product | eBioscience (551459) | Abcam (ab27988) | Dako (S330130-2) | |
| Clone | 1A8 | AE1/1E3 | NA | |
| Dilution | 1/200 | 1/100 | Ready to use | |
| Duration | ON, 4°C | RT, 1 hr | 1 min | |
| Secondary species | Rat | Mouse | NA | |
| AEC | 6 min | 22 min | NA |
Abbreviations: BD Bio, BD Biosciences; CST, Cell Signaling Technology; NA, not applicable; ON, overnight; RT, room temperature.