Table 2.

Details of IHC antibodies.

Antibody	Clone	Isotype /host	Supplier cat#	Dilution/Ab reaction	Antigen retrieval	Ab diluent	Position in sequential IHC/AEC reaction time
CD45	2B11+ PD7/26	IgG1/Mouse monoclonal	Dako Cat#M0701	1:50/1 h RT	EDTA (Ph8) water bath 100°C, 40 min	PBS + 1%BSA + 0,02% sodium azide	2°/30 min
CD4	EPR6855	IgG/rabbit monoclonal	Abcam Cat#ab133616	1:100/1 h RT	EDTA (Ph8) water bath 100°C, 40 min	PBS + 1%BSA + 0,02% sodium azide	4°/30 min
	4B12	IgG1/mouse monoclonal	Dako Cat#M7310	1:100/1 h RT	TRIS EDTA (Ph9) water bath 98.5°C, 20 min	Ventana Antibody Diluent Cat#251-018
CD8	4B11	IgG2b/mouse monoclonal	Thermo scientific Cat#MA1-80231	1:40/1 h RT	EDTA (Ph8) water bath 100°C, 40 min	PBS+1%BSA+ 0,02% sodium azide	4°/30 min
	4B11	IgG2b/mouse monoclonal	Novocastra Cat#NCL-L-CD84B11	1:100/1 h RT	TRIS EDTA (Ph9) water bath 98.5°C, 20 min	Ventana Antibody Diluent Cat#251-018
Foxp3	SP97	IgG/rabbit monoclonal	Thermo Scientific Cat#MA5-16365	1:100/1 h RT	EDTA (Ph8) water bath 100°C, 40 min	PBS + 1%BSA + 0,02% sodium azide	2°/30 min
	236A/E7	IgG1/mouse monoclonal	Abcam Cat#ab20034	1:100/1 h RT	Citrate buffer (Ph6) water bath 98.5°C, 20 min	Ventana antibody diluent Cat#251-018
Granzyme B	GrB-7	IgG2a/mouse monoclonal	Merk/Millipore Cat#MAB3070	1:20/1 h RT	EDTA (Ph8) water bath 100°C, 40 min	Ventana Antibody Diluent Cat#251-018	1°/30 min
				1:20/1 h RT	Citrate buffer (Ph6) water bath 98.5°C, 20 min	Ventana Antibody Diluent Cat#251-018
PDL1	SP142	IgG/rabbit monoclonal	Spring Bioscience Cat#M4424	1:100/1 h RT	EDTA (Ph8) water bath 100°C, 40 min	PBS + 1%BSA + 0,02% sodium azide	1°/30 min
				1:40/1 h RT	Cell conditioning solution (CC1) ventana BenchMark Cat#950-124	Ventana antibody diluent Cat#251-018

Antibodies and conditions applied in the optimized sequential IHC protocol are indicated in bold.