Immunohistochemical Analysis of p62 in IPF Lungs

Control or IPF lung tissues were fixed and embedded in paraffin wax; tissue sections (4 µm) were processed and stained as previously described^{18 (link)}. Briefly, the tissue sections were de-waxed, rehydrated and incubated with 3% hydrogen peroxide in methanol for 10 min to block endogenous peroxidase activity. Sections were then blocked with normal goat serum and incubated at room temperature with a primary antibody against p62/SQSTM1 (1:100, Progen, GP62_C), followed by a biotinylated secondary antibody (1:500, Vector Laboratories Ltd., UK); antibody binding was detected using streptavidin-conjugated horse-radish peroxidase and visualised using DAB (DAKO) before counterstaining with Mayer’s Haematoxylin. For H/E stain, Shandon Varistain 24-4 automatic slide stainer (Thermo Fisher Scientific) was used. For tinctorial stain, Trichrome stain (Abcam ab150686) was used according to the manufacturers’ instructions. Images were acquired using an Olympus Dotslide Scanner VS110.