Reaction buffer additive

The tertiary AC spheres were treated with 5-ethynyl-2-deoxyuridine (EdU, 200 ng/mL, Sigma) for 48 h as previously reported [28 (link),29 (link),34 (link)]. The spheres were fixed by 4% paraformaldehyde at room temperature for 10 min, followed by incubation in Click-iT reaction buffer, CuSO4, Alexa Fluor 555, and reaction buffer additive (Invitrogen) for 30 min. All nuclei were labeled with Hoechst 33342 (nuclei marker). The sphere samples were observed and imaged using Leica SPE confocal microscopy.

EdU (5-ethynyl-2’-deoxyuridine) staining was used to identify newly-generated cells. The EdU (Invitrogen) was delivered to each mouse weekly at 10 mg/kg by intraperitoneal injection from 6 weeks to 8 weeks after BCAS. The mice were sacrificed 2 months after BCAS and the brain was cut into frozen slices. Then the slices were fixed in 4% paraformaldehyde in PBS for 15 min. Next, the slices were washed twice with 3% BSA and permeabilized with 0.5% Triton X-100 in PBS for 20 min. The slices were again washed twice with 3% BSA in PBS and then incubated with a cocktail containing Click-iT™ reaction buffer, CuSO₄, Alexa Fluor^® 647 Azide, and reaction buffer additive (Invitrogen) for 30 min. The slices were then washed twice with PBS and further immunofluorescence staining was applied before using a laser scanning confocal microscope (FV3000, Olympus). All steps were carried out at room temperature.