Anti map2

Primary neurons were treated with a solution of MitoTracker Red CMXRos (Beyotime C1035 200 nM, Shanghai, China) for 17 min at 37 °C. Cells were then fixed with 4% PFA for 10 min at RT and blocked in PBS containing 10% NGS and 0.3% Triton X-100 for 30 min at RT. Cells were incubated in the following primary antibodies overnight at 4 °C: anti-LC3B (83506, Cell Signaling Technology), anti-Parkin (AF7680; Beyotime), and anti-MAP2 (8707, Cell Signaling Technology). Cells were incubated in the following secondary antibodies for 1 h at RT: goat anti-mouse Alexa Fluor 647 (A32728, Invitrogen) and goat anti-rabbit Alexa Fluor 647 (A32733, Invitrogen). Images were taken with a Leica TCS SP8 confocal laser scanning system.

Cells were collected and lysed in RIPA buffer containing protease inhibitor cocktail. After quantification, 20 μg of total proteins were used to perform an SDS-PAGE and WB analysis. PVDF membranes were incubated overnight with the primary antibodies (anti-Map2, Cell Signaling, #4542; anti-SK1 and anti-SK2, ECM Biosciences, #SP1621 and #SP4621, respectively; anti-S1P₂, Proteintech, 21180-1-AP; anti-NeuroD1, Abcam, ab60704; anti-β2-AR sc-9042, anti-β3-AR sc-13108 and anti-β-actin sc-1615, Santa Cruz Biotechnology) at 4 °C and then with specific secondary antibodies for 1 h at room temperature. Binding of the antibodies with the specific proteins has been detected by using Clarity Western ECL Substrate (Biorad).

Primary hippocampal neurons fixed in 10% buffered formalin were blocked in 10% goat serum for 1 h, then incubated with anti-Bassoon (1:10, Santa Cruz Biotechnology, Dallas, TX, USA), anti-synaptophysin (1:10, Santa Cruz Biotechnology, Dallas, TX, USA), anti-MAP2 (1:100, Cell Signaling Technology, Danvers, MA, USA) antibodies overnight at 4 °C. Cells were washed and incubated with Alexa-fluor 488 antibody or Alexa-568 antibody (1:200 dilution; Invitrogen, Molecular Probes, Carlsbad, CA, USA) for 1 h at room temperature and mounted on glass slides. Images were taken with a Zeiss Axiovert A1 microscope and processed using AxioVision 4.9.

2,5-HD (purity > 99%) and Hoechst 33342 were purchased from Sigma-Aldrich Co. LLC. (St. Louis, Missouri, USA). Rabbit polyclonal anti-NGF, anti-MAP2, anti-Akt, anti-p-Akt (ser 473), anti-p-Bad (ser 136) and mouse polyclonal anti-Bad were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit polyclonal anti-Bcl-xL, mouse polyclonal anti-cytochrome c and VDAC were purchased from Abcam Inc. (Cambridge, MA, USA). Goat polyclonal anti-Choline O-acetyltransferase (ChAT) was purchased from Millipore, (Bedford, MA, USA). Mouse monoclonal anti-NGF was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Alexa-Fluor 594 conjugated donkey anti-rabbit IgG, Alexa-Fluor 594 conjugated donkey anti-goat IgG, Alexa-Fluor 488 conjugated goat anti-mouse IgG and Alexa-Fluor 488 conjugated donkey anti-mouse IgG were purchased from Jackson ImmunoResearch (West Grove, PA, USA). Mouse polyclonal anti-β-actin, goat-anti-rabbit horseradish peroxidase (HRP)-conjugated IgG and goat-anti-mouse horseradish peroxidase (HRP)-conjugated IgG were purchased from ZSGB Biotechnology, Inc. (Beijing, China). RIPA lysis buffer, BCA Protein assay Kit, ECL enhanced chemiluminescence kit were purchased from Beyotime Biotechnology, Inc. (Shanghai, China). All other chemicals were of the highest grade commercially available.

Detection of newly synthesized proteins by PLA was performed as previously described (5 (link), 10 (link), 45 (link)). Immunostaining using mouse antipuromycin (Kerafast; 1:500) antibody in combination with rabbit anti–β-actin (Abcam; 1:1,000), rabbit anti–PSD-95 (cell signaling technologies; 1:1,000), or rabbit anti-Camk2a (Thermo; 1:1,000) was performed overnight at 4 °C. Following 5× PBS washes, PLA was performed (Sigma). Rabbit PLA^plus and mouse PLA^minus probes were used. PLA was performed according to the manufacture’s guidelines. Following PLA, anti-Map2 immunostaining (guinea pig anti-Map2, Cell Signaling; 1:5,000) was performed to label dendrites. Samples were imaged using a 40× oil objective on a LSM780 or LSM880. Z-stacks (0.43 μm) spanned the entire volume of imaged neurons. Images were analyzed using ImageJ. A 100-μm segment of the dendrite was assessed for the number of Puro-PLA puncta and the density of signal was calculated.

Primary NSCs and neurons were plated on poly-D-lysine-coated six-well plate for 7 days. After conditioned medium treatment, the medium was decanted and the cells were washed three times with PBS; they were then fixed with 4% paraformaldehyde for 30 min. Then NSCs and neurons were permeabilized with 0.3% Triton X-100 in PBS for 15 min. After being blocked in 3% bovine serum albumin (BSA) with PBS at room temperature for 1 h, NSCs and neurons were washed again in PBS and finally incubated overnight at 4°C with one of anti-MAP 2, anti-nestin, or anti-cleaved caspase-3 antibodies (1 : 200, Cell Signaling Technology, USA). Cells were then washed with PBS and incubated in one of two secondary antibodies (TRITC-anti-rabbit (555 nm) and DyLight 488-anti-rabbit 1 : 400, Cell Signaling Technology, USA) for 2 h. A 10 min incubation in DAPI was used to counterstain nuclei. Finally, fluorescent images were captured on an epifluorescence microscope (Leica, Germany), and fluorescence intensity was measured with ImageJ software.