Ab228549

For immunohistofluorescence staining, rats were anesthetized and transcardially perfused with 200 mL 0.9% saline, followed by 400 mL 4% paraformaldehyde at seven days post-transplantation (Supplementary Fig. 3a). Thereafter, the spinal cord was dissected (1.0 cm above and below the injured site, Supplementary Fig. 3b) and cryoprotected in 30% sucrose in 0.1 M PBS dehydrate at 4 °C. The tissues were sliced at 10 mm thickness by cryostat and fixed on the PLL-coated slides. The details of immunohistofluorescence staining were consistent with the above-mentioned description (Fan et al. 2019 (link)). The primary antibodies included anti-p75 (ab245134, 1:200, Abcam), GFP (ab6673,1:200, Abcam), Iba1 (019-19741, 1:500, Wako), iNOS (ab49999, 1:200, Abcam), Arg-1 (ab60176, 1:200, Abcam), NF68 (2835s, 1:50, Cell Signaling Technology) and DAPI (ab228549, 1:1000, Abcam). Other following procedures were same as the immunocytofluorescence.

For immunostaining, cells were fixed in 4% PFA for 10 min at room temperature. Cells were permeabilized (0.3% Triton X100—PBS for 10 min); blocked for 45 min with 5% donkey serum in 0.01% Triton X100—PBS and incubated with the primary antibodies: rabbit anti-ASCL1 1:200 (Abcam, ab74065), mouse anti-TUBB3, 1:1000 (Covance MMS-435P), chicken anti-TUBB3, 1:1000 (Abcam ab41489) mouse anti-MAP2 1:200 (Sigma, M4403), rabbit anti-MKI67, 1:1000 (Abcam, ab15580). The secondary antibodies (1:800, Invitrogen) were incubated for two hours at room temperature. Cells were then stained with 1 μg/mL DAPI (Abcam ab228549) for 20 min at room temperature. Images were acquired with a confocal microscope (Zeiss invert LSM510 or SP5 Leica).

Liver sections (5 μm) were dewaxed firstly, and treated with antigen retrieval solution (P0088, Beyotime) according to the manufacture’s protocol. Then the sections were blocked using Immunol Staining Blocking Buffer (P0102, Beyotime) at room temperature for 60 min. The fluorescence sections were incubated with primary antibody at room temperature for 60 min in a dark chamber, incubated with fluorescence labeling secondary antibody at room temperature for 60 min in a dark chamber, and then incubated with DAPI (ab228549, Abcam) for 30 min in a dark chamber. CD137⁺ cells were marked by green fluorescent signals. The sections were observed under an EVOS FL autofluorescence microscope (Thermo Fisher Scientific Inc.), and positive cells were automatically counted by Image-pro Plus 6.0 (Media Cybernetics).

BxPC3 cells were transfected with TF‐LC3 (mRFP‐GFP‐tandem fluorescent LC3) plasmid (#21074, addgene) by using lipofectamine 3000 (Invitrogen) and seeded on coverslips at 10000/well before receiving umbelliprenin or DMSO treatment. After added umbelliprenin or DMSO for 24 h, the cells were washed with PBS and fixed with 4% paraformaldehyde and stained with DAPI (ab228549, abcam). The green and red puncta were imaged by Zeiss LSM 700 confocal microscope equipped with a 63× oil immersion objective (Zeiss GmbH).

Neonatal mice were sacrificed by decapitation, and adult mice were sacrificed by CO₂ inhalation. Inner ears were dissected under operating binoculars and fixed in 4% PFA for 2 h at room temp. After fixation, inner ears were washed in PBS and stored in 4°C until dissection. Inner ears of mice older than P10 were decalcified in 0.25 M EDTA until entirely soft. The organ of Corti was dissected in PBS under operating binoculars. Specimens were permeabilized and blocked in 2% Triton X‐100 and 10% normal goat serum for 2 h at room temperature, and were then incubated overnight at 4°C in the appropriate primary antibody diluted in Phosphate Green antibody diluent (Bar Naor Ltd) according to manufacturer instructions. Specimens were washed and incubated for 2 h at room temperature in the relevant secondary antibody diluted in PBS according to manufacturer instructions and then mounted in ProLong Gold Antifade Mountant (Thermo Fisher Scientific) and imaged using a Zeiss LSM 880 (Zeiss, Oberkochen, Germany) equipped with an Airyscan detector. Antibody and stain concentrations were as follows: rabbit polyclonal myosin VIIa (Proteus Biosciences 25‐6790) 1:250, mouse anti‐FLAG (Sigma F3165) 1:1,000, DAPI (Abcam ab228549) 1:1,000, goat anti‐mouse (Abcam ab150119) 1:250, and goat anti‐rabbit Alexa Fluor 488 (Cell Signaling 4412s).