(A) PLISH experimental workflow. After an initial probe hybridization and enzymatic amplification step, up to five distinct channels can be simultaneously detected and imaged by conventional fluorescence microscopy, enabling direct visualization of RNA abundance. (B) PLISH detects single RNA molecules with single-cell resolution in tissues. PLISH staining for Foxj1 (white) and Scgb1a1 (red) in the bronchial epithelium of mouse lung shows a single ciliated cell (Foxj1+, arrowhead and asterisk) between Club cells (Scgb1a1+) in a planar view (top) and with orthogonal reconstruction (bottom). Note the discrete white puncta in the ciliated cell, which correspond to single Foxj1 transcripts. Dashed lines indicate the lateral and solid lines indicate the basal surface of airways. Foxj1, forkhead box J1; Scgb1a1, secretoglobin family 1A member 1; Scale bars, 10 μm. (C) Simultaneous RNA and protein detection in FFPE sections. FFPE human lung co-stained by PLISH (SCGB1A1, red) and indirect immunohistochemistry (anti-KRT5, grey) shows the expected localization of Club cells (SCGB1A1+, arrow) and basal cells (KRT5+, arrowhead) along the bronchial (Br) epithelium. Solid lines indicate the basal surface of airways. FFPE, formalin-fixed, paraffin-embedded; KRT5, keratin 5; SCGB1A1, secretoglobin family 1A member 1; Scale bar, 5 μm. (D) Discrimination of AT2 cells from macrophages by visual inspection of RNA abundance. PLISH staining in mouse lung for Lyz2 (purple) and Sftpc (green) allows clear discrimination of alveolar macrophages (Lyz2+ Sftpc-, white arrow) from AT2 cells (Sftpc+ Lyz2+, yellow arrow). AT2, Alveolar epithelial type II; Lyz2, lysozyme 2; Mac, macrophage; Sftpc, surfactant protein C; Scale bar, 20 μm. (E) Discrimination of AT2 cells from BASC cells by visual inspection of RNA abundance. PLISH staining for the Club cell marker (Scgb1a1, red) and AT2 cell marker (Sftpc, green) shows AT2 (Sftpc+), Club (Scgb1a1+) and BASC (Sftpc+ Scgb1a1Lo) cells. Note the discrete red puncta in the 'BASC' cells, which correspond to single Scgb1a1 transcripts. The cell types localize appropriately, with the AT2 cell in an alveolus (white arrow), the Club cells in the terminal bronchiole, and the double-positive BASCs at the bronchioalveolar junction (yellow arrows). Dashed lines demarcate the airway. AT2, alveolar epithelial type II; BASC, bronchioalveolar stem cell; Scgb1a1, secretoglobin family 1A member 1; Sftpc, surfactant protein C; Scale bar, 20 μm. (F) PLISH in patient tissue samples for molecular analysis of human disease. PLISH staining for SFTPC (green) in non-IPF human lung (left) marks AT2 cells (white arrow) distributed within alveolar septae (dashed lines). The adjacent panels show a magnified image of healthy cuboidal AT2 cells (dashed circles). PLISH staining in IPF human lung (right) shows densely cellular regions with architectural distortion of alveolar septae (dashed lines). SFTPCHi AT2 cells are inappropriately clustered (white arrowheads) and have abnormal flattened morphologies, as seen at higher magnification in right panels (dashed ellipses). AT2, alveolar epithelial type II; IPF, Idiopathic Pulmonary Fibrosis; SFTPC, surfactant protein C; Scale bar, 100 μm.