Immunohistochemical Analysis of Tumor-Infiltrating Immune Cells

Paraffin-embedded tissue blocks from patient tumours prepared for routine clinical histopathology were obtained from Sahlgrenska University Hospital. Sections (3–4 μm) from paraffin blocks were placed on glass slides and treated in Dako PT-Link using EnVision™ FLEX Target Retrieval Solution (high pH). The following primary antibodies were used: anti-CD3 (Dako/Agilent Technologies, Santa Clara, CA, USA; IR503), anti-CD4 (Dako; 4B12), anti-CD8 (Dako; C8/144B), anti-NKp46 (R&D Systems, Minneapolis, MN, USA; 195314), anti-granzyme B (Novocastra/Leica Biosystems, Wetzlar, Germany; 11F1), anti-PD-L1 kit (Dako; PD-L1 IHC 28-8 pharmDx; according to manufacturer’s instructions), and anti-Ki-67 (Dako; MIB-1). Immunohistochemical staining was performed in a Dako Autostainer Link using EnVision™ FLEX according to the manufacturer’s instructions (DakoCytomation). EnVision™ FLEX+ (LINKER) mouse was used for anti-NKp46, anti-CD25, anti-granzyme B, and anti-Ki-67. Positive and negative controls were included in each run. Each staining was evaluated by a board-certified pathologist (O.N.). The Ki67 index was calculated by manually counting the percentage of labelled tumour cell nuclei on printed images [28 (link)].

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Hofving T., Liang F., Karlsson J., Yrlid U., Nilsson J.A., Nilsson O, & Nilsson L.M. (2021). The Microenvironment of Small Intestinal Neuroendocrine Tumours Contains Lymphocytes Capable of Recognition and Activation after Expansion. Cancers, 13(17), 4305.

Publication 2021

EnVision™ FLEX Target Retrieval Solution anti-CD3 anti-CD4 anti-CD8 anti-NKp46 anti-Ki-67

Anti cd3 Antibodies Cd25 Granzyme b Mib 1 Mouse Nkp46 Nuclei of cell Paraffin Pathologist Patient Pd l1 Tissue Tumour

Corresponding Organization : University of Western Australia

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Variable analysis

independent variables

Paraffin-embedded tissue blocks from patient tumours

dependent variables

Immunohistochemical staining for CD3, CD4, CD8, NKp46, granzyme B, PD-L1, and Ki-67
Ki67 index (percentage of labelled tumour cell nuclei)

control variables

Sections (3–4 μm) from paraffin blocks were placed on glass slides
Dako PT-Link using EnVision™ FLEX Target Retrieval Solution (high pH) for sample preparation
Dako Autostainer Link using EnVision™ FLEX for immunohistochemical staining
EnVision™ FLEX+ (LINKER) mouse for specific antibodies (anti-NKp46, anti-CD25, anti-granzyme B, and anti-Ki-67)
Positive and negative controls included in each run

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