Envision hrp rabbit mouse polymer

Formalin-fixated paraffin-embedded blocks of renal tumors were analyzed by immunohistochemistry. Heat-induced epitope retrieval was performed with EDTA for CD3 and with Trilogy for PD-L1, PD-1 and CD68. Staining was performed on a DAKO autostainer plus. After blocking endogenous peroxidase, sections were incubated for 45 min with rabbit monoclonal anti-PD-L1 antibody (1:1000; Cell Signaling #13,684, clone E1L3N), mouse monoclonal anti-CD68 antibody (1:100; Dako #M0876, clone PG-M1), mouse monoclonal anti-CD3 antibody (1:50; Dako #M7254, clone F7.2.38) and rabbit polyclonal anti-PD-1 antibody (1:150; Zytomed #516-18,662). Sections were washed and incubated with Dako REAL EnVision HRP Rabbit/Mouse polymer, which reacts with DAB-Chromogen, according to the manufacturer’s protocol. Different pathologic scores were determined [24 (link)–26 (link)], including tumor cell staining score (TPS-score), immune cell staining (IC-score) and a combined score (CPS-score) for PD-L1 and the immune cell staining scores (IC-score) for PD-1, CD3 and CD68, respectively. Total RNA was extracted from slices cut in line from the immunohistochemistry blocks (High Pure FFPE RNA Micro Kit, Roche Diagnostics GmbH, Germany).

For immunohistochemistry, heat‐induced epitope retrieval was performed with EDTA for LN‐511 subunit LAMA5, COL1 and COL4 and with Trilogy for BCAM. Staining was performed on a DAKO autostainer plus. After blocking endogenous peroxidase, sections were incubated for 45 min with mouse monoclonal anti‐BCAM antibody (1:25; R&D systems #MAB1481, clone 87207), mouse monoclonal anti‐LAMA5 antibody (1:50; Atlas Antibodies # AMAb91124, clone CL3118), mouse monoclonal anti‐Collagen IV antibody (1:100; Dako/Agilent; Waldbronn, Germany; #M0785, clone CIV22) or rabbit polyclonal anti‐Collagen I antibody (1:200; Abcam #ab34710). Sections were washed and incubated with Dako REAL EnVision HRP Rabbit/Mouse polymer, which reacts with DAB‐Chromogen, according to the manufacturer´s protocol.