Table 1

Final, optimized six-plex mIF assay conditions for characterizing the PD-1/PD-L1 axis

Staining order	1° antibody	Clone	Concentration* (µg/mL)	Vendor	Incubation (min)	2° HRP	TSA-Opal	Lot number	Dilution
1	PD-L1	E1L3N	5.83	CST	30	PowerVision	520	2 581 789	1:150
2	CD8	4B11	N/A†	Life Technologies	30	Opal Polymer	540	2 566 905	1:300
3	FoxP3	236A/E7	0.5	Abcam	60	PowerVision	570	2 553 339	1:150
4	CD68	PGM-1	300	Dako	60	PowerVision	620	2 567 659	1:150
5	PD-1	EPR4877(2)	4.97	Abcam	60	PowerVision	650	2 566 920	1:150
6	CK	AE1/AE3	0.8	Novus Biologicals	30	Opal Polymer	690	2 556 626	1:150

*All antibodies were diluted using Akoya’s antibody diluent/blocking buffer.
†This antibody is not purified and is supplied as crude tissue culture supernatant. As such, the antibody concentration is not available.