Cd68 clone kp1

All histological sections were reviewed, and 4-µm sections were obtained from the most representative formalin-fixed paraffin-embedded (FFPE) tissue blocks. Immunohistochemical stains (IHC) were performed utilizing antibodies directed against CD3 (clone LN10, dilution 1/250, Leica Biosystems, UK), CD4 (clone SP35, Ready to Use Predilute Antibody, Ventana, USA), CD8 (clone C8, dilution 1/250, Dako, Denmark), CD20 (clone L26, dilution 1/300, Dako, Denmark), and CD68 (clone KP1, dilution 1/1500, Dako, Denmark) utilizing clinically validated protocols. Slides were scanned at ×40 magnification on the Aperio GT450 brightfield instrument (Leica Biosystems). The resolution of the images was 0.26 µm/pixel at ×40. The images were 24-bit contiguous standard pyramid tiled TIFFs compressed via JPEG with a quality setting of 91. A board-certified neuropathologist selected and annotated regions for analysis using Aperio ImageScope Software (Leica Biosystems). The annotated regions of each stained slide were analyzed using proprietary nuclear and cytoplasmic algorithms. Cells within each region of interest were graded based on intensity of staining (0, 1 + , 2+ or 3 + ). Cells with an intensity of 1+ or higher were considered positive for immunostaining markers. Immunohistochemistry (IHC) scores were expressed as a percentage of positive cells (0 to 100) within the region of interest.

Immuno-histochemistry was performed on frozen sections stained with the primary antibodies for a-SMA (clone 1A4; Dako, Glostrup, Denmark), CD68 (clone KP1, Dako), uncarboxylated and carboxylated MGP (ucMGP and cMGP, respectively; 1:25; IDS, Boldon, UK), BMP-2 (1:20; Genetics Institute, Cambridge, MA) and Osteocalcin (1:50; Anawa Trading, Wangen, Zürich, Switzerland). Secondary antibodies used were Biotinylated sheep anti-mouse IgG (1:250; Amersham, Little Chalfont, Buckinghamshire, UK) or sheep anti-rabbit IgG (1:1000, Dako). Antibodies were visualized by alkaline phosphatase–coupled avidin-biotin complex (Dako), in combination with red alkaline substrate kit I (Vector SK-5100; Vector Laboratories, Burlingame, CA); nuclei were counterstained with hematoxylin. Furthermore, all samples we routinely stained for Hematoxylin Eosin (HE), von Kossa, oil red O and Picro-Sirius red.

Twenty-three formalin-fixed paraffin-embedded human HNSCC samples were stained using the Opal 7-Color Automation IHC Kit-50 Slide according to manufacturer’s instructions (Akoya Biosciences) using the following antibodies and antigen retrieval processes: with antibodies against PD-L1 (clone E1L3N, Cell Signaling Technologies cat. 13684S, Opal 620), CD11c (clone 5D11, Sigma Cell Marque cat. 111M-15, Opal 650), CD68 (clone KP1, Dako cat. M0814, Opal 690), and pan-CK (clone AE1/AE3, Dako cat. M351501-2, Opal 540) on a Bond RX autostainer (Leica Biosystems). Slides were dewaxed (Leica), heat treated in ER2 or ER1 antigen retrieval buffer depending on the antibody for 20 min at 93 °C (Leica), blocked in Antibody (Ab) Diluent (Akoya Biosciences), incubated for 30 min with the primary Ab, 10 min with horseradish peroxidase (HRP)-conjugated secondary polymer (anti-rabbit and anti-mouse, Akoya Biosciences), and 10 min with HRP-reactive OPAL fluorescent reagents (Akoya Biosciences). Slides were washed between staining steps with Bond Wash (Leica) and stripped between each round of staining with heat treatment in antigen retrieval buffer. After the final heat treatment in antigen retrieval buffer, the slides were stained with spectral DAPI (Akoya Biosciences), and coverslipped with Prolong Diamond mounting media (Thermo Fisher).

Five‐micrometer‐thick paraffin‐embedded archival lung tissue from deceased patients with SARS‐CoV‐2 positive pneumonia was stained on the Bond III automated staining platform (Leica, Wetzlar, DE). After deparaffinization and Ag retrieval, slides were incubated with Bond Peroxide Block for 5 min. Samples were then incubated with the primary monoclonal mouse anti‐human CD68 (clone KP1, dilution 1:7000; Dako, Santa Clara, CA, USA) and with the primary monoclonal MS4A4A (HPA029323, dilution 1:500; Sigma), followed by signal amplification and visualization using the Leica Bond Polymer Refine Detection Kit (Leica, Wetzlar, DE). Operating parameters for application of the detection system reagents were followed as suggested and incorporated into the software by Leica Biosystems. Finally, the slides were counterstained with hematoxylin, dehydrated and cover‐slipped.