Ab5543

Primary antibodies for rabbit anti-OPTN (ab23666) and rabbit anti-beclin-1 (ab62557) were purchased from abcam (Cambridge, MA, USA); mouse anti-LC3 was purchased from MBL International Corporation (M152-3, Woburn, MA, USA); mouse anti-alpha-synuclein was purchased from BD Transduction Labs (610786, San Jose, CA, USA); chicken anti-MAP2 (AB5543), sheep anti-tyrosine hydroxylase (AB1542), anti-tryptophan hydroxylase (AB1541), and anti-choline acetyltransferase (AB1582) were purchased from EMD Millipore (Billerica, MA, USA). Normal donkey serum (017-000-121) and secondary antibodies, Alexa Fluor^®; 647 anti-sheep (713-605-147), Alexa Fluor^®; 488 anti-rabbit (711-545-152), and Cy^TM3 anti-mouse (715-165-151) were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). Rotenone (R8875) and glycerol (G5516) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Triton X-100 was purchased from Fisher BioReagents (BP151-500, Fair Lawn, NJ).

For immunostainings, fixed neurons were permeabilized with 0.2% Triton X-100 and incubated with PBS based blocking solution containing 5% goat serum, 1% bovine serum albumin (BSA), 0.1% gelatin, 0.1% Triton X-100, and 0.05% Tween 20. Primary antibodies [guinea pig anti-synaptophysin (1:2000; 101004, Synaptic systems), mouse anti-flag (1:1000; F1804, Sigma), rabbit anti-myc (1:1000; 06-549, Millipore), chicken anti-MAP2 (1:8000; AB5543, Millipore), and mouse anti-HA (1:1000; MMS-101R, Covance)] were diluted in a solution containing 1% BSA and 0.1% gelatin. The following secondary antibodies were used: goat anti-mouse Alexa Fluor 647 (1:2000; A21236, Life Technologies), goat anti-rabbit Alexa Fluor 647 (1:2000; A21245, Life Technologies), goat anti-guinea pig Alexa Fluor 568 (1:2000; A11075, Life Technologies), and goat anti-chicken Alexa Fluor 405 (1:500; ab175674, Abcam). The stained samples were mounted on microscope slides using Prolong Gold antifade reagent (P36934, Life technologies). Confocal images were acquired using a LSM Zeiss 710 confocal microscope (alpha Plan-Apochromat 63×/1.46 OilKorr M27 objective).

To verify the effects of the hNR1 antibody on somatic Npas4 expression, cortical neurons 14–17 DIV were incubated with 1 µg/ml of hNR1 antibody for 2, 4, or 24 h. Alternatively, to induce expression of Npas4, neurons were treated with NMDA (2 μm, Tocris) for 2 h. Untreated cells were used as a control. To study the effect of hNR1 and NMDAR antagonist (2R)-amino-5-phosphonovaleric acid (AP5) on NMDA-induced Npas4 expression, NMDA treatment was preceded by 6 h or 24 h of hNR1 (1 µg/ml) or 3 h of AP5 (100 μm, Tocris) treatment. After treatment, cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min and washed 3 × 5 min with PBS. Subsequently, cells were permeabilized and blocked with a solution containing 5% normal goat serum, 2% BSA, and 0.1% Triton in PBS, for 1 h. Neurons were then incubated with primary antibodies against Npas4 (1:1500, rabbit, Activity Signaling) and MAP2 (1:2000, chicken, Millipore, AB5543) in antibody solution containing 2% BSA in PBS for 2 h at room temperature (RT). Cells were then washed 3 × 5 min with PBS and incubated with the differently labeled secondary antibodies (1:1000 in antibody solution, Invitrogen, Thermo Fisher Scientific), for 2 h at RT and washed 3 × 5 min with PBS. Finally, coverslips were dipped in H₂O and mounted in Pro-Long Diamon Antifade Mountant (Thermo Fisher Scientific).