Dab reagent

IHC was performed to evaluate the fibrotic phenotype of lung tissues. After deparaffinization and hydration, the slices were heated with the antigen retrieval solution for 10 min. Then, the slices were penetrated with PBS for 5 min followed with H₂O₂ treatment for about 15 min, and then blocked with goat serum (SL038, Solarbio) for 60 min at RT to eliminate the activity of endogenous peroxidase. The slices were incubated overnight at 4°C with antibodies against the following proteins: α-SMA (1:200, AF1032, Affinity) and fibroblast-specific protein 1 (FSP-1, 1: 200, A19109, Abclonal). The next day, the slices were incubated with HRP-labeled secondary antibody (1:500, #31460, ThermoFisher, USA) at 37°C for 60 min. After developed with DAB reagent (DA1010, Solarbio), the slices were then counterstained with hematoxylin (H8070, Solarbio). Finally, the slides were mounted and observed under a microscope.

The cells and tissues were fixed with a fixator, then embedded with paraffin, and finally sectioned in paraffin blocks. Paraffin sections were cleaned and dewaxed with ethanol solutions of different concentrations. Antigen repair solution was used to repair the antigen, and PBS was washed 3 times. Polyclonal rabbit anti-USP39 antibody (Abcam, US) was added and incubated overnight at 4°C, then washed with PBS solution. HRP-labeled secondary antibody (Abcam, US) was added and incubated at 4°C for 30 min: DAB dyeing with DAB reagent (Solarbio Life Science, Beijing), microscope observation, and photography.