Bca protein assay

After 4 h of ventilation treatment, the lungs were lavaged with 0.3 ml PBS by tracheal intubation and pumped back after 3 s. The same operation was repeated three times in a total volume of 0.9 ml PBS, and the recovery rate was 90%. The lungs were blotted dry with filter paper, weighed to obtain the wet weight, and then were dried in an oven at 65°C for 48 h to obtain the dry weight. The lung wet/dry (W/D) ratio was calculated. Protein concentrations were determined using the BCA protein assay (ComWin Biotech, China). myeloperoxidase (MPO) activity, an indicator of the neutrophils and macrophages infiltration, was determined using test kits obtained from Nanjing Jiancheng Bioengineering Institute, China. The enzymatic activity was assessed by measuring the changes in the absorbance at 450 nm using a Varioskan Flash multifunction plate reader (Thermo Scientific, IL, United States).

Normal lymph node (NLN) and MLN tissues were homogenised and lysed in cold RIPA buffer containing protease inhibitor cocktail. Then, the lysates were centrifuged for 15 min. The supernatants were collected, and the protein concentration was analysed by BCA protein assay (Beijing ComWin Biotech). The samples were incubated with anti-fibronectin polyclonal antibodies (Abcam) or anti-β-actin antibodies at 4 °C overnight. The secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H + L) was used after washing three times in TBST. After incubation for another 1 h and washing, ECL reagents were added, and then Western blot images were captured.