Alexa fluor 647 goat anti rabbit

Cells seeded on the scaffolds were allowed to grow for 24 h followed by washing with PBS, and were subsequently fixed with 4% paraformaldehyde for 10 min. The fixed cells were then permeabilised for 20 min using 0.5% Triton X-100 (Scharlau, Barcelona, Spain) before blocking by immersion in 10% goat serum (Elabscience, Houston, TX, USA) for 1 h at 37 °C. HDF was incubated with the primary antibodies, rabbit anti-human collagen I antibody (1:300 dilution; Abcam, Cambridge, MA, USA) and mouse anti-alpha smooth muscle antibody (1:500 dilution; Abcam, Cambridge, MA, USA) overnight at 4 °C, followed by secondary antibody incubation with Alexa Fluor 647 goat anti-rabbit (Abcam) and Alexa Fluor 488 goat anti-mouse antibodies (Abcam), both diluted to 1:1000, for 2 h at 37 °C. The cells were counterstained with DAPI (Thermo Fisher, Waltham, MA, USA) for 15 min to visualise the nuclei. Between each post-fixing step, the samples were washed with PBS containing 0.1% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA). Images were captured using an A1R confocal laser scanning microscope (Nikon, Minato City, Japan).

Before immunofluorescent staining, the deparaffinization process was performed to remove the penetrated paraffin from the tissue. The slides were immersed in a sodium citrate buffer at pH 6.0 and heat samples near boiling in a water bath for 10 min. Sections were then permeabilized by treating with 0.25% Triton X-100 (30 min, room temperature), and blocked for 1h by 0.5% BSA (Sigma, Germany) with 5% NGS. Sections were then exposed overnight with the diluted primary antibodies, including mouse anti-nestin (1:50; Abcam, UK) for neural stem/progenitor cells, mouse anti-GFAP (1:100; Sigma, Germany) for astrocytes, and rabbit anti-doublecortin (1:100; Santa Cruz, Germany) for immature neurons at 4 °C and rinsed in TBS three-time before the application of secondary antibody. Alexa Fluor 647 goat anti-mouse (1:600; Abcam, UK) and Alexa Fluor 647-goat anti-rabbit (1:500; Abcam, UK) were applied for 60–90 min at room temperature. In negative controls, the primary antibodies were omitted. The slides were then washed with TBS and nuclei were stained with DAPI (Sigma-Aldrich, Germany). The sections mounted with glycerol buffer under coverslips. The percentages of nestin, GFAP, and doublecortin positive cells (GFP-positive cells) per image field were calculated relative to the total number of GFP-positive cells in hNS/PCs and hNS/PCs + PM groups.

Cells were washed twice in 2 mL ice-cold PBS and harvested with a rubber policeman after being lysed on ice for 20 min in lysis buffer (Beyotime, Shanghai, China). Equal amounts of total protein were solubilized by sodium dodecyl sulfate (SDS) sample buffer (Beyotime, Shanghai, China), separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). Membranes were incubated with corresponding polyclonal and secondary antibodies. Signal was detected with a Super ECL Reagent substrate (Hai Gene, Harbin, China). Source and product number of antibody: LC3B (Abcam, ab48394), SQSTM1/p62 (Abcam, ab101266), Beclin 1 (Bioworld, AP0769), p-ULK1 (Affinity Biosciences, AF4387), ULK1 (Proteintech, 20986-1-AP), p-ATG13 (Bioworld, BZ40743), ATG13 (Bioworld, bs6045), ATG5 (Proteintech, 10181-2-AP), p-PI3K (CST, 4228), PI3K (CST, 4249), p-PTEN (CST, 9554), PTEN (CST, 9188), p-Akt (CST, 13038), Akt (CST, 4691), p-mTOR (Abcam, ab84400), mTOR (CST, 2972S), p-p70 S6K (CST, 9205), p70 S6K (CST, 2708), p-4E-BP1 (CST, 2855), 4E-BP1 (CST, 9644), GAPDH (Bioworld, AP0066), HRP-linked antibody (anti-rabbit IgG, CST, 7074), Goat Anti-Rabbit (Alexa Fluor®488) (Abcam, ab150077), goat anti-rabbit (Alexa Fluor®647) (Abcam, ab150079).

For single fluorescent labeling, samples were blocked with 5% normal serum and incubated overnight at 4°C with rabbit anti–h-αS (1:3000). For double fluorescence, sections were first incubated overnight with either rabbit anti-RFP (1:3000) or mouse anti–Syn-O2 (1:2000; TAB-0748CLV, Creative Biolabs) and then incubated with anti–h-αS. Labeling of these primary antibodies was achieved using a secondary antibody conjugated with DyLight 488 or DyLight 594 (1:300; Vector Laboratories). Sections were rinsed, mounted on coated slides, and coverslipped with Vectamount mounting medium (Vector Laboratories). For triple fluorescence, tissue sections were processed using the following sequential labeling procedures. First, for detection of mCherry-conjugated hM3D, mCherry was labeled by incubations with rabbit anti-RFP (1:3000), donkey anti-rabbit Fab fragment (1:200; Jackson ImmunoResearch), and goat anti-donkey Alexa Fluor 594 (1:300; Abcam). Second, SOD2 was labeled by incubations with rabbit anti-SOD2 (1:1000) and goat anti-rabbit Alexa Fluor 647 (1:300, Abcam). Last, h-αS was labeled by overnight incubation with rabbit anti–h-αS conjugated with Alexa Fluor 488 (1:400; ab216124, Abcam). Fluorescence images were collected on Zeiss microscopes (LSM700, LSM800, LSM880, or LSM900) using the ZEN software (Carl Zeiss).

To examine autophagosome–lysosome fusion, an immunocolocalization assay between LC3 (an autophagosome membrane marker) and LAMP‐2 (a lysosome membrane marker) in pancreas was conducted. Paraffin‐embedded pancreatic tissue sections (5 μm) were dewaxed, rehydrated and incubated with 5% H₂O₂ in methanol for 10 min. to quench the endogenous peroxidase. After high‐pressure antigen retrieval, the sections were blocked with 5% bovine serum albumin for 1 hr and incubated with the primary antibodies overnight at 4°C. On the following day, secondary goat antimouse Alexa Fluor^® 488 (1:100) and goat antirabbit Alexa Fluor^® 647 (1:100) antibodies (Abcam, Cambridge, UK) were applied for 1 hr. Finally, the sections were covered with mounting medium (Vector Laboratories, Burlingame, CA, USA) and observed under a confocal laser scanning microscope (LSM‐510; Carl Zeiss, Oberkochen, Germany). With the ImageJ 1.48v software (National Institutes of Health, Bethesda, MD, USA), the percentage of LAMP‐2 stained area that colocalized with LC3 [yellow area/(yellow area + free red area) × 100%] per high power field in the merged image was calculated.