Cross sections (10 μm) were cut with a cryostat (CM1900, Leica Microsystems, Nussloch, Germany) at -20 ºC. The sections were air-dried and fixed with 4% paraformaldehyde (v/v) in 0.1 M phosphate-buffered saline (pH 7.5)43 (link),63 (link),64 (link).
Interstitial fibrosis was evaluated by Masson-trichrome staining using the Accustain Trichrome Stain Kit (#HT15-1KT; Sigma-Aldrich) in accordance with the manufacturer’s protocol as described previously22 (link). Interstitial fibrotic regions were quantified using Image J 1.48v software (National Institute of Health, Bethesda, MD, USA) to determine the percentage of blue-stained area in the Masson-trichrome sections22 (link),43 (link),64 (link). DNA fragmentation was determined by TUNEL staining using an Apoptosis in situ Detection Kit (#293-71501; Wako, Osaka, Japan). The total number of TUNEL-positive nuclei was counted manually in six sections of three groups (Control (n = 4), BO (n = 5), and Pro + BP (n = 4)) over a microscopic field of 20 x, averaged, and expressed as the ratio of TUNEL-positive nuclei (%)20 (link),22 (link),43 (link),64 (link).
Interstitial fibrosis was evaluated by Masson-trichrome staining using the Accustain Trichrome Stain Kit (#HT15-1KT; Sigma-Aldrich) in accordance with the manufacturer’s protocol as described previously22 (link). Interstitial fibrotic regions were quantified using Image J 1.48v software (National Institute of Health, Bethesda, MD, USA) to determine the percentage of blue-stained area in the Masson-trichrome sections22 (link),43 (link),64 (link). DNA fragmentation was determined by TUNEL staining using an Apoptosis in situ Detection Kit (#293-71501; Wako, Osaka, Japan). The total number of TUNEL-positive nuclei was counted manually in six sections of three groups (Control (n = 4), BO (n = 5), and Pro + BP (n = 4)) over a microscopic field of 20 x, averaged, and expressed as the ratio of TUNEL-positive nuclei (%)20 (link),22 (link),43 (link),64 (link).
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