Alexa fluor 555 or 488 secondary antibody

The lungs were collected and sectioned at the thickness of 4 μm. The slides were stained with H&E (Beyotime Institute of Biotechnology, Haimen, China) at room temperature. Microscopic lung fibrosis scoring was done using the Ashcroft scale^{33 (link)}. Microscopic lung injury scoring was done using a novel acute lung injury scoring system^{34 (link)}. The mean score of five fields was recorded as the final score of the mouse. Every group included 10 mice, and two pathologists were blind to the study design.
Masson-Trichrome staining was done using a commercial kit (Beyotime Institute of Biotechnology, Haimen, China) according to the instructions. ECM deposition was analysed using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA). Immunofluorescence was used to locate and evaluate renin expression and ECM deposition. The antibodies and their concentrations were as follows: anti-renin (1:100 dilution; Santa Cruz), anti-fibronectin (1:200 dilution; Abcam), anti-collagen I (1:200 dilution; Abcam) and anti-α-SMA (1:200 dilution; EMD Millipore), and these were subsequently conjugated with Alexa Fluor 555 or 488 secondary antibodies (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The antigens were visualized using a Leica DFC425 fluorescence microscope [Leica Microsystems (Schweiz) AG, Heerbrugg, Switzerland].

Immunofluorescent staining was performed on paraffin vertical sections using antibodies against ATG7 and LC3II. Briefly, the placental vertical sections were dewaxed in xylene, rehydrated and heated in a microwave with citrate buffer (pH = 6.0) for antigen retrieval before exposure to the primary antibodies. Then, sections were immersed in 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase. Unspecific immunoreactions were blocked using 5% inactivated goat serum in PBS for 30 minutes at room temperature. The sections were incubated with ATG7 (1:500, Sigma, USA) or LC3II (1:200, Cell Signaling Technology, USA) antibodies overnight at 4°C. Sections were washed extensively and incubated with the corresponding Alexa Fluor 555 or 488 secondary antibody (1:1000; Invitrogen, Waltham, MA, USA) and DAPI (5 μg/ml, Invitrogen) at room temperature for 2 hours in a dark box.