Alexa fluor 488

The immunofluorescence (IF) assay was performed on RAW264.7 cells as described previously [12 (link), 48 (link)]. Briefly, the primary antibodies against PCNA (1:50 dilution, Proteintech, USA), Rab7 (1:50 dilution, Proteintech, USA), LAMP1 (1:50 dilution, Proteintech, USA), LC 3 (1:100 dilution, Cell Signaling Technology, USA) and secondary antibodies conjugated with Alexa Fluor 488 and Alexa Fluor 594 dyes (Cat. no. ZF-0513, ZF-0511, and ZF-0512; ZSGB-BIO, China) were used for the test. To observe the superstructures of F-actin, phalloidine conjugated with Alexa Fluor 594 (Sigma, USA) was used to stain the cells for 1 hour at room temperature. The morphology of cell nuclei was shown by DAPI (Sigma, USA) staining for 10 min. The imaging experiments were digitized on laser scanning confocal microscopes (LSM700, Zeiss, Jena, Germany) as described previously [12 (link), 48 (link)]. In addition, the z-stacks plug-in was used to perform the consecutive scans of the cells in the direction of Z-axis.

BMSCs (P3) were inoculated in 12-well plates at a cell density of 1 × 10⁴ cells/well. ADPN (10 μg/ml) and the control group without drugs were added accordingly. The medium was replaced every other day for 7 days. The wells were rinsed with PBS three times. Four percent neutral paraformaldehyde was added. Fifteen minutes later, 0.1% Triton X-100 was applied to lysate the cells for 15 min. Five percent goat serum was used for blocking. Drops of primary antibodies were added (the same antibodies as in animal experiments) with dilution concentrations of 1:100 and incubated overnight in a wet box in a refrigerator at 4°C. Secondary antibody (antibody dilution: 1:200, ZSGB-Bio, China Beijing, Alexa Fluor^® 488, ZF-0512, Alexa Fluor^® 594 ZF-0513) was added, and the nuclei were stained with DAPI.

IFAs were performed as the standard protocols. Briefly, cells were previously seeded into a 24-well plate and infected with the virus at a multiplicity of infection (MOI) of 0.01. At 36 h postinfection, the culture supernatants were removed, and the cells were fixed with ice-cold acetone and incubated with monoclonal antibody (2A10) against flavivirus E protein at 37°C for 1 h (34 (link)). After washing three times in phosphate-buffered saline (PBS), cells were incubated with the second antibody conjugated with Alexa fluor 488 (ZSGB-BIO) for 45 min. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen), and the positive cells were examined under a fluorescence microscope (Zeiss).

For the in vitro assay, scaffolds were washed with PBS, blocked with 3% bovine serum albumin (BSA) for 30 min, and incubated overnight at 4°C with primary antibodies against IGFBP3 (1:50, rabbit anti‐human, Santa Cruz) in addition to 2U of Rhodamine Phalloidin (Biotium, Hayward, CA); DAPI was used to stain nuclei.
For the in vivo assay, the scaffolds, retrieved at 7 days in vivo, were embedded in optimal cutting temperature compound, and snap frozen at −20°C. Sections (8 µm thick) were incubated overnight at 4°C with primary antibodies against Sca‐1 (1:500, 7 H4 L3, Invitrogen, CA) and PDGFR‐α (1:500, Invitrogen). Secondary antibody labeled with Alexa Fluor 488 (1:100, donkey anti‐rabbit) or Cy3 (1:100, goat anti‐rat, ZSGB‐BIO, Beijing, China) were used as appropriate. DAPI was used to stain nuclei. The number of Sca‐1⁺ PDGFR‐α⁺ cells were counted and the percentage of Sca‐1⁺ PDGFR‐α⁺ cells to the total cells were determined Fluorescence images were acquired using a Two Photon Laser Scanning System (LSM 510 NLO, Zeiss, Oberkochen, Germany). A total of three images per animal distributed within the defect area, with 800× magnification, were analyzed.

The scaffolds retrieved at 7 days in vivo were embedded in optimal cutting temperature compound, and snap frozen at –20 °C. Sections (8-μm thick) were held overnight at 4 °C with primary antibodies against Sca-1 (1:500, 7 H4L3; Invitrogen, CA, USA) and PDGFR-α (1:500; Invitrogen) [25 (link)]. As appropriate, secondary antibodies labeled with Alexa Fluor 488 (1:100, donkey anti-rabbit) or Cy3 (1:100, goat anti-rat; ZSGB-BIO, Beijing, China) were used, and DAPI was used to stain nuclei. Fluorescence images were acquired using a Two Photon Laser Scanning System (LSM 510 NLO; Zeiss, Oberkochen, Germany). Endogenous cells and Sca-1⁺PDGFR-α⁺ MSCs migrating into the defect site were quantified at day 7 based on immunofluorescent images. A total of three images per animal distributed within the defect area, with 800× magnification, were analyzed.

Oocytes were collected and washed with PBS supplemented with 0.1% PVA and then fixed with 4% PFA at RT for 30 min. The oocytes were then permeabilized with 1% Triton-100 in PBS at RT for 8–12 h and blocked with 1% BSA for 1 h at RT. After washing with 0.05% Tween-20 containing 0.1% Triton-100 in PBS, the oocytes were incubated with anti-α-tubulin FITC antibodies (Thermo Fisher Scientific, USA) at a dilution of 1:200, and an anti-SIRT1 antibody (Santa Cruz, CA, USA) at a dilution of 1:200 overnight at 4 °C. After washing in 0.05% Tween-20 containing 0.1% Triton-100 in PBS, the oocytes were stained with Alexa Fluor 488 (ZSGB-BIO, Beijing, China) secondary antibody for 1 h at RT. Oocytes were incubated in 10 μg/ml propidium iodide (PI) at RT to view the nucleus. After several washes, the oocytes were mounted on glass slides, and images were captured under a confocal laser scanning microscope (LAS - Leica TCS-SP8, Germany) with the same scanning settings.