Immunofluorescence Analysis of Lung Cells

Immunofluorescence analysis of cells and lung tissues was performed as described previously 35 (link). For this, the following primary antibodies were employed: rat anti-PDGFRα, mouse anti-α-SMA, rabbit anti-PDGFRα, rabbit anti-PTPN13, and rabbit anti-NOX4. Alexa Fluor 488-conjugated goat anti-mouse antibody and Alexa Fluor 594-conjugated goat anti-rabbit antibody (Invitrogen no. A-11001 and A-11037, Carlsbad, CA, 1:200 dilution) were used as secondary antibodies. Nuclei were stained with DAPI (Sigma no. D9542). The images were observed under an FV3000 confocal laser scanning microscope (Olympus, Tokyo, Japan). For quantification analysis, five high-power fields were analyzed in lung sections taken from each mouse. We then determined the percentages of PDGFRα⁺, PTPN13⁺, and NOX4⁺ cells in total α-SMA⁺ cells.