Mice were sacrificed at various time points after bleomycin treatment under anesthesia. The trachea was cannulated, and the left lungs were inflated with 0.5 mL of 10% neutral buffered formalin. Mouse lung tissue was fixed, embedded in paraffin, sectioned to 5 μm slices for Masson’s trichrome13 (link)49 (link) and BMPER staining. Anti-BMPER (ab75183, 1:200, Abcam) and anti-rabbit HRP-DAB cell and tissue staining kit were used according to the manufacturer’s instructions (R&D systems, Minneapolis, MN, USA). The semiquantitative Ashcroft score was used to score pulmonary fibrosis50 (link). In short, upon 200× magnification, each successive field was given a score ranging from 0 (normal) to 8 (total fibrous obliteration of the field). All scores from 5 sections were averaged41 (link).
Paraffin-embedded sections of normal and IPF lungs were used for immunofluorescence staining. Antibodies specific for BMPER (ab75183, 1:200, Abcam) and α-SMA (F3777, 1:250, Sigma, St. Louis, MO, USA) were used for staining. Alexa Fluor-546-conjugated secondary antibody was from Life Technologies. The nucleus were labeled with DAPI (Vector lab, Burlingame, CA, USA), photographed with a Leica TCS SP5 confocal microscope, and analyzed with Leica confocal software.
Paraffin-embedded sections of normal and IPF lungs were used for immunofluorescence staining. Antibodies specific for BMPER (ab75183, 1:200, Abcam) and α-SMA (F3777, 1:250, Sigma, St. Louis, MO, USA) were used for staining. Alexa Fluor-546-conjugated secondary antibody was from Life Technologies. The nucleus were labeled with DAPI (Vector lab, Burlingame, CA, USA), photographed with a Leica TCS SP5 confocal microscope, and analyzed with Leica confocal software.
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