A11031

For in situ hybridization of miR‐124, double‐digoxigenin locked nucleic acid probe (RNO‐MIR‐124‐3P: CATTCACCGCGTGCCTTA, Tm: 84°C, 339111 YD00614870‐BGC, miRCURY LNA™ miRNA Detection Probe, Qiagen) was used at a final concentration of 30 nM. Hybridization was performed according to the manufacturer's instructions. Digoxigenin was revealed with a tyramide‐based method (1:50, TSATM ‐Plus Fluorescein System, NEL741001KT, PerkinElmer). Pan neuronal staining was also processed (monoclonal mouse antibodies, 1:500, Neuro‐ChromTM Pan Neuronal Marker Millipore MAB 2300) and revealed with a goat anti‐mouse antibody conjugated to Alexa 568 (1:500, A11031, Thermo Fisher). Nuclei were stained with DAPI (DAPI Fluoromount‐G^®, SouthernBiotech).

Primary antibodies used were: mouse-IgG1 anti-gp91^phox (D162-3, MBL), rabbit serum anti-SNAP23 (111202, Synaptic Systems), rabbit serum anti-stx8 (110083, Synaptic Systems), rabbit serum anti-stx7 (110072, Synaptic Systems), mouse-IgG1 anti-stx7 (sc-514157, Santa Cruz), rabbit serum anti-stx12 (299022, Synaptic Systems), rabbit serum anti-VAMP8 (104302, Synaptic Systems), rabbit serum anti-Vti1b (164002, Synaptic Systems), rabbit serum anti-stx5 (110053, Synaptic Systems), rabbit serum anti-LAMP1 (L1418, Sigma), rabbit serum anti-TGN38 (sc-27680, Santa Cruz), mouse-IgG1 anti-EEA1 (610456, BD Biosciences), rabbit serum anti-p67phox (07-002, Merck Millipore), rabbit monoclonal-IgG anti-GAPDH (2118, Cell Signaling Technology), mouse-IgG1 anti-FITC (200-602-037, Jackson ImmunoResearch Laboratories) and mouse-IgG1 isotype control (400102, Biolegend) antibodies. The following secondary antibodies were used for immunofluorescence: goat anti-mouse IgG (H+L) Alexa-Fluor-488 or -568-conjugated (A-11029 and A-11031, ThermoFisher) and goat anti-rabbit IgG (H+L) Alexa-Fluor-568 or -647-conjugated (A-11036 and A-21245, ThermoFisher) antibodies. For immunoblotting, we used the secondary antibodies goat anti-rabbit or anti-mouse IgG (H+L) IRDye-800CW-conjugated (926-32211, Li-Cor) antibodies, and for flow cytometry, goat anti-mouse IgG (H+L) Alexa-Fluor-488-conjugated antibodies were used.

Drp1 KO MEFs were plated on 0.1% gelatin (G1393, Sigma) coated coverslips in 12-well plates. 24 hr after plating, the cells were transfected with the vectors expressing the indicated Myc-tagged Drp1 constructs by using the TransIT^®–2020 transfection reagent (MIR 5404, Mirus). 24 hr after transfection, the cells were fixed with 4% PFA in PBS for 20 min at room temperature before 5 min permeabilization with 0.1% Triton-X-100. Cells were then incubated with blocking buffer (5% normal goat serum (31872, Thermofisher Scientific), 0.05% Triton-X-100 in PBS) for 1 hr at room temperature. Primary antibodies against Myc (Drp1) and Tom20 (mitochondria) were incubated overnight at 4°C. The secondary Alexa-488-labeled goat anti-rabbit (A11034, ThermoFisher Scientific) and Alexa-568-labeled goat anti-mouse (A11031, ThermoFisher Scientific) antibodies were incubated for 1 hr at room temperature. The expression of Myc-tagged Drp1 and mitochondrial morphology were examined by using anti-Myc (sc-40, Santa Cruz), and anti-Tom20 (11802–1-AP, Proteintech) antibodies, respectively. Confocal images were obtained using a 60X oil-immersion objective mounted on an Olympus Fluoview 1000 or 3000 confocal microscope and analyzed by Fiji-ImageJ (NIH).

Hippocampal neurons were fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich) in phosphate-buffered saline (PBS) for 20 min, permeabilized in 0.1% Triton-X100 in PBS for 5 min, and blocked in 3% bovine serum albumin (BSA, Calbiochem, San Diego, CA, USA) in PBS for 1 h at 37 °C. They were then incubated with primary antibodies in 3% BSA in PBS overnight at 4 °C and followed by secondary antibodies for 1 h at 37 °C, and mounted in Fluoromount-G (Electron Microscopy Sciences, Hatfield, PA, USA). Antibodies were as follows: anti-HA (rabbit, ab9110-100, 1:1000; Abcam Cambridge, MA, USA), anti-Myc (mouse, 9E10, 1:500; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), goat anti-rabbit Alexa-Fluor 568 (A11036, 1:500; Thermo Fisher Scientific) and goat anti-mouse Alexa-Fluor 568 (A11031; 1:500; Thermo Fisher Scientific).

Immunostaining was performed as described previously [12 (link)]. In brief, after treatment, cells were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized for 15 min with 0.2% Triton X-100 (Merck Millipore, 94101-L), and then blocked with 5% goat serum for 1 h. they were subsequently incubated with primary antibodies overnight at 4 °C followed by incubation for 1 h with secondary antibodies at room temperature. Their nuclei were stained with a DAPI solution. Images were captured with a fluorescent microscope (Nikon Eclipse 80i equipped with Nikon PLAN FLUOR × 40 objective) or Nikon confocal microscope (Nikon, N-STORM and A1). Photographic images were resized and analyzed by ImageJ software. The following primary antibodies were used for immunostaining: SQSTM1 (Santa Cruz Biotechnology, sc-28359); K48-linked Ub chain-specific antibody (Merck Millipore, 05-1307); FLAG Tag (Prospec, ANT-146); Phospho-SQSTM1 (S403)-specific antibody (Cell Signaling Technology, 39786), Alexa Flour 488- or 568-conjugated secondary antibodies (Thermo Fisher Scientific, A11034, A11029, A11031, A11036); See Additional file 1: Table S2 for further details and dilutions of all antibodies.