Lsm fv1000

hNPCs were cultured on laminin-coated 13 mm glass coverslips, and the cells were fixed post HG treatment using 4% paraformaldehyde (PFA) for 18 min at room temperature, and then blocked with 5% goat serum for 1 h. Subsequently, the cells were incubated at 4 °C overnight with rabbit anti-SLIT1 (1:100, ab129345, Abcam), mouse anti-ROBO2 (1:200, sc-376177, Santa Cruz Biotechnology), mouse anti-SRGAP1 (1:200, sc-81939, Santa Cruz Biotechnology), mouse anti-YAP (1:50, sc-101199, Santa Cruz Biotechnology), rabbit anti- TAZ (1:50, ab84927, Abcam), rabbit anti-CDC42 (1:50, #2462, Cell Signaling Technology), mouse anti-Nestin (1:250, ab18102, Abcam), mouse anti-SOX-2 (1:250, ab171380, Abcam) or rabbit anti-Musashi-1 (1:500, ab52865, Abcam). The following day, the coverslips were incubated with secondary antibody for 1 h at room temperature. The nucleus was counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) for 5 min, and the coverslips were mounted with fluorescence mounting media (DAKO, Cat. no. S3023) onto glass slides. Images were taken using a confocal microscope, LSM FV1000 (Olympus).

Forty-thousand–60,000 BV2 microglial cells were seeded on poly-lysine coated coverslips in 24-well culture plates. Following transfection and LPS treatment, the cells were fixed with 4% PF, washed and blocked with 5% goat serum followed by incubation with the following antibodies: anti-CAMK2A antibody (mouse monoclonal antibody, 1:200, Cat. No. MA1-048, Thermo Fisher Scientific) and anti-BDNF antibody (rabbit recombinant monoclonal antibody, 1:200, Cat. No. ab108319, Abcam) overnight at 4°C. The cells were then incubated with secondary Rb-Cy3 antibody (1:200, Cat. No. C2306, Sigma-Aldrich) or Ms-Cy3 (1:200, Cat. No. C2181, Sigma-Aldrich) and lectin, a microglia specific marker (1:200, L0401, Sigma-Aldrich), followed by counterstaining with DAPI. The coverslips then mounted with fluorescent mounting medium (DakoCytomation, Glostrup, Denmark). Slides were allowed to dry for at least 1 day before imaging. Images were taken using LSM FV1000 (Olympus).

HeLa cells stably transduced with V5-tagged SMSr expression constructs were seeded on glass coverslips. Cells were fixed using 4% (w/v) paraformaldehyde in PBS at RT and quenched using 25 mM NH₄Cl in PBS. Cells were permeabilized using PBS containing 0.1% (w/v) saponin and 0.2% (w/v) BSA. Coverslips were immunostained using mouse anti-V5, sheep anti-TGN46, rabbit anti-calnexin primary antibodies followed by donkey anti-rabbit Cy5, donkey anti-mouse Cy3, and donkey anti-sheep/goat FITC antibodies. Coverslips were mounted with Prolong Gold Antifade Reagent (Thermo Fisher Scientific). Images were captured using a confocal microscope (Olympus LSM FV1000) equipped with two spectral and a single standard detector, an UPLSAPO 60x/NA 1.35 oil immersion objective (Olympus) and an Olympus laser box with AOTF laser combiner. Fluorophores were excited using 488, 559, and 635 nm lasers. Excitation light was reflected by a 405/488/559/635nm dichroic mirror. Emitted light was collected using secondary dichroic mirrors SDM-560 and SDM-640 and a barrier filter BA 655-755 for FITC, Cy3, and Cy5 respectively. To avoid cross-talk between image channels, images for different fluorophores were collected sequentially in a descending order of wavelength, i.e. longer to shorter wavelengths. Image analysis was performed using ImageJ (NIH) software.

The cell slides were incubated overnight at 4 °C with the primary antibody to EP300 (ab275378, 1:200, Abcam), followed by incubation with secondary antibody Cy3-conjugated donkey anti-rabbit IgG (C2571, 1:100; Sigma-Aldrich) for 120 min at ambient temperature. After that, the slides were incubated with the antibody to SMYD2 (ab234862, 1:200, Abcam) for 4 h at room temperature, followed by incubation with secondary antibody FITC-conjugated donkey anti-goat IgG (F3512, 1:100; Sigma-Aldrich) for 2 h at room temperature. After nuclei counter-staining with DAPI (Vector Laboratories, Burlingame, CA, USA), fluorescent mounting medium (Dako Cytomation, Glostrop, Denmark) was used to mount the slides. All images were taken under a confocal microscope (LSM FV 1000; Olympus).

Nd(NO₃)₃·6H₂O and Gd(NO₃)₃·6H₂O were purchased from Beijing HWRK Co., LTD (Beijing, China). NH₄VO₃ was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Dopamine hydrochloride and polyethylene glycol (PEG) were acquired from Sigma-Aldrich Co. (Saint Louis, MO, USA). Dimethyl sulfoxide (DMSO) was obtained from Xilong Chemical Co., Ltd. (Swatow, China). Bovine serum albumin (BSA) was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). All chemicals were used as received.
TEM images of NPs were acquired from a FEI Tecnai G2 S-Twin operating at an acceleration voltage of 200 kV. The X-ray powder diffraction was obtained using a D8 Advanced diffractometer (Bruker) with Cu Kα radiation (λ = 0.15405 nm). The UV–VIS absorption was recorded on U-3100 spectrophotometer (Hitachi). The Zeta potential was measured using a Malvern instrument Zetasizer Nano. The Confocal laser scanning microscopy images was performed on a LSM FV 1000 instrument (Olympus). Thermogravimetry data were recorded on a Netzsch Thermal Analyzer (STA 409) in an ambient environment with a heating rate of 10 °C min⁻¹ from room temperature to 700 °C.