Ab150075

The cells grown on slides were fixed with 4% paraformaldehyde for 15 min and blocked with 1× phosphate buffer saline supplemented with 3 mg/mL bovine serum albumin, 100 mM glycine, and 0.25% Triton X-100 for 30 min. Subsequently, the slides were incubated with primary antibody rabbit anti-BDNF (1:500, ab108319), rabbit anti-light chain 3II (LC3II) (1:1000, ab48394), mouse anti-Tau (Tau-13, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-BRUCE (5 µg/mL, ab19609) at 4 °C, followed by culture with the combination of the fluorophore and Alexa Fluor^® 647 secondary antibody (1:200, ab150075) at room temperature for 1 h. All the antibodies used above were provided by Abcam except Tau. Then, the cell slides were added with 4′ 6-diamidino-2-phenylindole for nucleus staining, then immersed in distilled water, dried and observed under a fluorescence microscope (Zeiss, Thornwood, NY) or FV-1000 confocal microscope.
For morphological analysis, the cells were fixed with 4% paraformaldehyde for 15 min and incubated with tau antibody (1:200, ab64193, Abcam) for 1 h, followed by another incubation with fluorophore combined with Alexa Fluor^® 647 secondary antibody (1:200, ab150075) for 1 h. The cells were then observed under the fluorescence microscope, and the length of the main axon in each cell was measured by five independent experiments.

BMMSCs in 24-well plates were fixed with 4% paraformaldehyde (Aladdin, Shanghai, China), followed by permeabilization with 0.1% Triton X-100 (Beyotime) for 10 min. After blocking for 30 min, the cells were incubated with antibodies at 4°C for 8 h. Subsequently, the cells were incubated with a secondary antibody (ab150075, Abcam, Cambridge, UK) conjugated with phalloidin (Beyotime). Finally, the nucleus was counterstained with DAPI (Beyotime), and images were captured using a fluorescence microscope (Zeiss).

Separate staining for DNase1 and DNase1L3 was performed using the protocol described previously [73 (link)]. Sections were incubated overnight at 4 °C with DNase1-polyclonal antibody (BS-7651R, Bioss, Woburn, MA, USA, 0.01 µg/mL) or DNase1L3-polyclonal antibody (BS-7653R, Bioss, Woburn, MA, USA, 0.01 µg/mL). The next day, sections were incubated for 30 min at room temperature with a donkey anti-rabbit-IgG Alexa Fluor 647 (AB150075, Abcam, Cambridge, UK) and DAPI (Invitrogen, Grand Island, NY, USA). Images were captured using a fluorescence microscope (KEYENCE BZ-9000, Keyence Corporation, Osaka, Japan). Exposure time was calibrated using the isotype control antibody (AB37415, Abcam, Cambridge, UK), with adjustments made to achieve a negative fluorescence signal in the control. Three to five samples were inspected microscopically, and one representative image was taken per condition.