Bond rx fully automated research stainer

In situ hybridization of human FFPE meningeal sections was performed with a Bond-Rx Fully Automated Research Stainer (Leica Biosystems) following the RNAScope LS Multiplex Fluorescent Reagent Kit (322800, ACD) User Manual for BDZ 11 with the following modifications. Sections were baked for 30 min at 60 °C prior to dewaxing for 30 s at 72 °C. Sections were antigen retrieved using HIER with a pH 6.0 citrate buffer solution (AR9961, Leica Biosystems) for 20 min at 100 °C, washed, and treated with ACD Protease III for 15 min at 40 °C prior to probe hybridization for 2 h at 40 °C. TSA Plus fluorescein, Cy-3, and Cy-5 (Akoya Biosciences) were diluted 1:500 for signal development. Autofluorescence due to age-related endogenous lipofuscin was quenched with a 30-second exposure to True-Black Lipofuscin Autofluorescence Quencher (Biotium), and slides were washed with PBS before mounting. Imaging was performed on either a CellInsight CX7 LZR High Content Analysis Platform (ThermoFisher Scientific) or an Eclipse Ti2-E microscope (Nikon) at ×20 and ×40.

4 μm sections were mounted onto saline‐coated glass slides to ensure section adhesion through subsequent staining procedures. Slides were stained using the BOND RX Fully Automated Research Stainer (Leica Biosystems) using the Bond Polymer Refine Detection Method Algorithm as per manufacturer instructions (DS9800 Leica). The slides were incubated with a dilution of 1:200 rabbit monoclonal antibody against human anti‐FGF21 (FGF21 antibody [MA532652] size 100 UL, Invitrogen). Negative controls were carried out with rabbit serum diluted to the same concentration as the primary antibody. The extent of FGF21 immunostaining was assessed using ImageJ.

For the multiplexed immunohistochemical staining of the sections, we used BOND RX Fully Automated Research Stainer (cat: 21.2821; Leica Biosystems) and an Opal Polaris 7Color IHC Detection Kit (cat: P-000003, Akoya Biosciences). All procedures were performed according to the manufacturer’s instructions. In summary, deparaffinized sections were incubated with citrate- or Tris-based antigen unmasking solutions (for heat-induced epitope retrieval) at 98°C for 20 min. They were then treated with hydrogen peroxide and a protein-blocking reagent to prevent the nonspecific binding of antibodies to the sections. Sections were sequentially treated with the primary antibodies, horseradish peroxidase (HRP)-conjugated antibodies, and specific fluorophores to detect the proteins of interest. Multiple staining rounds were performed using the following anti-human antibodies: anti-AhR (cat: LS-C783005-100; LS Biosciences), anti-CD68 (cat: 76437; Cell Signaling Technology), anti-CD4 (cat: ab181724; Abcam), anti-CD8 (cat: CD8-4B11-L-CE; Leica Biosystems), anti-FoxP3 (cat: 98377; Cell Signaling Technology, anti-PanCK (cat: AE1/AE3-601-L-CE; Leica Biosystems). Tissue sections were counterstained with Spectral DAPI (4′,6-diamidino-2-phenylindole, cat: SKU FP1490; Akoya Biosciences).