Af0261

PCNA and CD68 were detected by immunohistochemistry (IHC), while insulin and glucagon were revealed by immunofluorescence staining with a previously described procedure^{28 (link),29 (link)}. Sections were incubated with the primary antibody, mouse anti-PCNA (AF0261, Beyotime, China), rabbit anti-CD68 (BA3638, Boster, China), goat anti-insulin (sc-7839, Santa Cruz, Santa Cruz, CA), or mouse anti-glucagon (BM1621, Boster, China) at 4 °C overnight, and with a secondary antibody, HRP-goat anti-mouse (Catalog#: 32230, Zymed, San Francisco, CA), HRP-goat anti-rabbit (A0208, Beyotime, China), donkey anti-goat cy3 (A0502, Beyotime, China), or goat anti-mouse FITC (A0568, Beyotime, China) for 1 h at room temperature. For IF, the nucleus was stained with DAPI (1 μg/ml, Dojindo, Japan) after washing with PBS. A total of 3 randomly chosen pancreatic sections from each mouse in each group (about 40 islets) were used for IHC or IF study. Finally, images were collected at equal exposure conditions and at the same magnification (20x objective lens) and analyzed by using the Image-Pro Plus 6.0 software.

The lung tissues or PASMCs were homogenized in RIPA lysis buffer (Beyotime, Shanghai, China) with a protease and phosphatase inhibitor cocktail (Beyotime, Shanghai, China). Samples containing 20–40 μg of protein were separated by 10% SDS-PAGE gel and then transferred to polyvinylidene fluoride (PVDF) membranes (G.E. Healthcare, Germany). After blocking with 5% skim milk or 5% BSA, the PVDF membranes were incubated overnight on a shaker at 4°C with the following primary antibodies: collagen1 (AF6524, 1:1,000, Beyotime), collagen3 (AF6531, 1:1,000, Beyotime), MMP2 (AF0234, 1:1,000, Beyotime), MMP9(AF5234, 1:1,000, Beyotime), PCNA (AF0261, 1:1,000, Beyotime), SRC (AF1831, 1:1,000, Beyotime), p-SRC (AF5923, 1:1,000, Beyotime), PIM1 (AF1807, 1:1,000, Beyotime), eNOS (AF6792, 1:1,000, Beyotime), and α-tubulin (sc-5286, 1:500, Santa Cruz). After incubation with horseradish peroxidase (HRP)-linked secondary antibody (Beyotime, Shanghai, China), the protein bands were imaged through Molecular Imager ChemiDoc XRS System (Bio-Rad, Philadelphia, USA). Densitometric quantification was performed by ImageJ (NIH, USA). The α-tubulin served as a loading control.

The hippocampus cells proliferation were performed by PCNA immunohistochemistry, in brief, tissue sections were incubated with PCNA mouse monoclonal antibody (0.2 μg/ml, AF0261, Beyotime, China) for 45 min and the results were observed by an optical microscope [30 (link)]. Meanwhile, hippocampus tissue sections were incubated with TUNEL reagent (Roche, Germany) for 60 min at 37 °C, and then stained with Hoechst 33,258 (1 μg/ml, Sigma-Aldrich, USA) for 20 min [31 (link)]. Afterward, the tissue sections were mounted with mounting medium (Applygen Technologies Inc., China) and visualized under a confocal microscope. Five visual fields were randomly selected, and the percentage of positive hippocampal cells was calculated as the apoptosis index.