Dako omnis immunostainer

Slides were stained for routine diagnostics, over a period of several years. Formalin‐fixed paraffin‐embedded blocks were cut into 3‐µm sections with a Leica RM2255 Automated Microtome (Leica Biosystems B.V., Amsterdam, the Netherlands). Sections were placed on microscope slides and dried at either 60°C for 30 min to 16 h, or at 37°C for 72 h. After being dried, the slides were deparaffinised, and antigen retrieval was performed in citrate buffer (Target Retrieval Solution, pH 6) for 40 min. Immunohistochemistry (IHC) was performed according to a laboratory‐developed test protocol. Slides were stained with the Dako Omnis immunostainer and Dako EnVision Flex+ reagents and 1:20 dilution of PD‐L1 clone 22C3 (Dako Omnis, Dako Agilent Technologies, Leuven, Belgium). The IHC slides were then counterstained with haematoxylin, and coverslips were applied. Tonsil and placental tissue were used as positive controls for PD‐L1 expression.

Cultured cells were fixed in 4% formaldehyde (Q Path, VWR Chemicals, Radnor, PA, USA) and prepared for paraffin embedding with the Epredia™ Cytoblock™ Cell Block Preparation System (ThermoFisher Scientific). Sections were stained with hematoxylin and eosin (H&E) using the Leica ST5020-CV5030 Stainer Integrated Workstation (Leica Biosystems, Amsterdam, The Netherlands). The H3F3A p.G34W immunohistochemical stain (RM263, RevMAb, Biosciences, San Francisco, CA, USA) was performed with the Dako Omnis immunostainer (Agilent, Santa Clara, CA, USA) according to the standard diagnostic procedure at LUMC.

After fixation for at least 24 hr in 10% neutral buffered formalin, the tissue samples were dehydrated in a graded series of ethanol and xylene, mounted in paraffin and cut into 3-μm-thick sections. In addition to hematoxylin and eosin (HE), Elastica-van-Gieson (EvG), Berlin Blue (Fe), periodic acid Schiff stain (PAS), Alcian Blue-periodic acid Schiff stain (abPAS), Giemsa, Gomori Trichrome, and Kongo Red stain were used by following routine protocols. For immunohistochemistry, the following antibodies were used: AE1/3 (Dako/IR053), TTF-1 (Dako/IR056), CK7 (Dako/IR619), CK5/6 (Dako/IR780), p40 (Zytomed/MSK097), Ki67 (Dako/IR626), CD68 (Dako/M0876), CD61 (Dako/M0753), CD31 (Dako/IR610), CD34 (Dako/IR623), ASMA (Dako/IR611), CD3 (Dako/IR503), CD20 (Dako/IR604), MUM1 (Dako/IR644), collagen IV (Dako/M0785), and tenascin (Chemicon/MAB19101). All immunostaining were performed with the Dako Omnis immunostainer (Agilent) by following routine procedures. The sections were examined microscopically (Axio Imager. M2, Carl Zeiss Microscopy GmbH), and representative photographs were obtained (Axiocam 506 color, Carl Zeiss Microscopy GmbH; ZEN 2.6 (blue edition), Carl Zeiss Microscopy GmbH).