Opal multiplex fluorescent immunohistochemistry system

For multiplex staining of RFX2 in lung tissue, 2-μm-thick paraffin sections of lungs were reacted with the following primary antibodies: rabbit anti–E-cadherin (#3195, Cell Signaling Technology, MA, USA), rabbit anti–SP-C (#HP9050, Hycult Biotech, Netherlands), and rabbit anti-RFX2 (#12622-1-AP, Proteintech). For detection, Histofine Simple Stain Mouse MAX-PO secondary antibody (Nichirei, Japan) was used with an Opal multiplex fluorescent immunohistochemistry system (Akoya Biosciences, MA, USA) that included Opal 520, Opal 570, Opal 650, 1× amplification diluent, and AR6 buffer. For immunohistochemistry of BMPR2, de-paraffinized sections were stained with mouse anti-BMPR2 (#GTX60415, GeneTex), and the substrate solution was reacted with Histofine SAB-PO (M) Kit (Nichirei, Japan). For contrast staining, hematoxylin solution TypeM, (Mutoh Chemical Industry, Japan) was used. Images of stained tissues were recorded with a BZ-X710 fluorescence microscope.

Each joint and lymph node was fixed in 4% paraformaldehyde/PBS, decalcified in 2.5% EDTA, and stored in 7% sucrose at 4 °C for one month. Paraffin Sects. (2 μm) were stained with hematoxylin and eosin (H&E; Muto, Tokyo, Japan) for immunohistochemical analysis. The sections were stained with the following primary antibodies: rat anti-F4/80 (Bio-Rad), rabbit anti-CD3 (Genemed Biotechnologies, South San Francisco, CA, USA), rat anti-PTPRC/CD45R (Aviva Systems Biology, San Diego, CA, USA), rabbit anti-syndecan 1 (Bioss Antibodies, Woburn, MA, USA), anti-CD4, and anti-CD8 (Cell Signaling Technology, Danvers, MA, USA). Histofine simple stain mouse MAX-PO secondary antibodies and the Opal multiplex fluorescent immunohistochemistry system (Akoya Biosciences, Marlborough, MA, USA) were used according to the manufacturers’ protocols. All images were captured using a fluorescence microscope (BZ-X710; Keyence, Osaka, Japan).

For mouse lung immunohistochemistry, the deparaffinized sections were stained with the following primary antibodies: rabbit anti-E-cadherin and rabbit anti- MMP7 (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-SP-C (Hycult Biotech, Uden, Netherlands), and ACE-2 (R&D Systems, Minneapolis, MN, USA). For human lung immunohistochemistry, deparaffinized sections were stained with the following primary antibodies: rabbit anti-E-cadherin (Cell Signaling Technology), rabbit anti-proSP-C (Merck, Darmstadt, Germany), rabbit anti-ACE-2 (R&D Systems), and mouse anti-Laminin γ2 N-terminal fragment (γ2pf, Funakoshi, Tokyo, Japan). Histofine simple stain mouse MAX-PO secondary antibodies (Nichirei, Tokyo, Japan) and the Opal multiplex fluorescent immunohistochemistry system (Akoya Biosciences, Marlborough, MA, USA) were used according to the manufacturer’s protocol. All the images were captured using a fluorescence microscope (BZ-X710; Keyence, Osaka, Japan). To calculate the percentage of Ace2⁺ cells in E-cadherin⁺ bronchioles or invasive epithelial cells from the UIP lungs of four mice, five images (200× magnification) were captured and the percentage of Ace2 positive cells was calculated by ImageJ Fiji.