Masson trichrome staining kit

C57BL/6 mice were acquired from Vital River Laboratories (Beijing, China) and primary mouse lung fibroblasts (MLF), MIC-CELL-0040 from PriCells (Hubei, China). RNA extraction kit was purchased from Tiangen Biotech (Beijing, China), reverse transcription kit and fluorescent quantitation PCR kit from Takara (Tokyo, Japan). Nrf2, Bach1, HO-1, GPx1 rabbit anti-mouse primary antibodies were purchased from Abcam (Cambridge, MA, USA). Protein Extraction Kit was obtained from Sigma-Aldrich (St. Louis, MO, USA) and ROS kit were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Enzyme linked immunosorbent assay (ELISA) Kit was obtained from R&D (R&D Systems, Minneapolis, MN, USA) and Cloud-Clone Corp (USCN Life Science Inc, Wuhan, China). Primers were synthesized by Sangon (Shanghai, China). Hematoxylin staining kit and Masson trichrome staining kit were purchased from Solarbio (Beijing, China). TGF-β1 recombinant protein was obtained from R&D, and PFD was obtained from Shionogi & Co., Ltd (Osaka, Japan).

The decalcified femoral heads of patients and rats were paraffin‐embedded and cut into 4‐μm‐thick sections using a microtome (Leica Microsystems, Wetzlar, Germany). The sections were stained with haematoxylin and eosin and a Masson trichrome staining kit (Solarbio Co., Ltd., Beijing, China). Images were acquired using an ECLIPSE80i microscope (Nikon, Tokyo, Japan), and the proportion of empty lacunae was determined.

For histological analysis, whole lungs were fixed for 24 h in 4% phosphate-buffered formalin (PFA), dehydrated and embedded in paraffin, and 5 μM sections were stained using a Masson trichrome staining kit (G1340, Solarbio, Beijing, China) following the manufacturer’s protocols. To develop a modified Ashcroft score, 5 fields in each lung section were randomly selected under 100× magnification to obtain the mean score by two pathologists in a blinded fashion [45 (link)]. For immunohistochemical staining of a-SMA and collagen I, tissue slides were dewaxed by dimethyl benzene, polarized with descending concentrations of alcohol, and rinsed with deionized water. Endogenous peroxidase activity was blocked by incubating sections in 3% hydrogen peroxide solution at room temperature for 30 min after antigen retrieval. The 10% normal goat serum was used to block the slides for 1 h. Primary antibodies were incubated overnight at 4 °C followed by incubation of secondary antibody for 60 min at room temperature. The slides were visualized by DAB staining, and counterstaining was performed with hematoxylin. Images of the stained sections were obtained with a panoramic scanner (3DHISTECH, Budapest, Hungar).