Synaptophysin

For immunofluorescence staining, 12-μm-thick coronal frozen sections were obtained from OCT embedded blocks (from−1.46 mm to−2.46 mm posterior to bregma). To block non-specific antibody binding, 10% normal goat serum was used after antigen retrieval with citrate buffer (0.01 M, pH 6.0) at 90°C for 15 min. Specific primary antibodies were applied at 4°C overnight as follows: IBA1 (Wako, Japan), GFAP (Sigma-Aldrich, USA), CD68 (Serotec, USA), cleaved caspase 3 (Cell Signaling Technology, USA), PSD95, and synaptophysin (Synaptic System, Germany). The sections were then incubated with specific Alexa Fluor 568 or 488 secondary antibodies (Invitrogen, USA) for 2 h at room temperature.
Apoptosis was detected using the TdT-mediated dUTP nick-end labeling (TUNEL) technique (In Situ Cell Death Detection Kit TMR red, Roche, USA). The nuclei were labeled with 4'-6-diamidino-2-phenylindole (DAPI) (3 μM, Sigma-Aldrich, USA). Photographs were observed under a laser scanning confocal fluorescent microscope (5×, 20×, and 40× oil immersion objectives) (Carl Zeiss LSM 900, Germany). Z-stack images were acquired at 1,024 × 1,024 resolution. All quantitative analyses were performed on at least three images acquired from three serial sections per animal.

Whole TA muscles collected from 20‐day‐old mice were fixed in 4% paraformaldehyde and permeabilized in 5% goat serum and 1% Triton X‐100 in PBS. Samples were then incubated in rabbit anti‐neurofilament (NF‐M, 1:100; Sigma) and synaptophysin (1:200; Synaptic Systems) antibodies overnight at 4 °C, followed by Alexa Fluor 488 goat anti‐rabbit IgG (H+L) (1:500; Life Technology) and rhodamine‐α‐bungarotoxin (α‐BT) (1:1000; Life Technology). Muscle fibres were teased and mounted using Hydromount mounting medium (National Diagnostics). A minimum of 100 NMJs from each sample were randomly selected and captured using confocal laser scanning microscopy. The areas of synapse end plates were measured using ImageJ software.

Primary antibodies: Vesicular Glutamate Transporter 1 (VGLUT1, Millipore, USA), Synaptophysin (Synaptic System, Germany), Synaptotagmin-2 lumenal domain (Synaptic System), detyrosinated α-tubulin (Glu-α-tubulin, Synaptic System), α-tubulin (Sigma Aldrich, Japan), Kinesin motor protein KIF1A (AbCam, USA), GluR1/2-Cy3 extracellular domain (BIOSS antibodies).
Secondary antibodies: AlexaFluor 405, 488, 568 and 647, DAPI, Quantum dots Q655 and Q585 (all from Life Technologies, USA). CypHer5E (C5E, Synaptic System).

Samples containing equal amounts of protein were denatured in SDS sample buffer, subjected to SDS-PAGE, transferred onto a PVDF membrane, and applied to immunoblot analysis. Protein bands on immunoblots were visualized by a chemiluminescence method (Millipore) and an imaging documentation system (ImageQuant LAS 4000, GE healthcare). Images were analyzed using ImageJ. Primary antibodies against CCNY (Proteintech group), GFP (Roche), GluA1 (a gift from Michael Ehlers, Pfizer Neuroscience), phospho-GluA1 (S845) (Thermo scientific), PSD-95 (Thermo scientific, 7E3-1B8), Synaptophysin (Synaptic Systems), Prox1 (Proteintech group), Ctip2 (Genetex), Py (a gift from D.T.S. Pak, Georgetown Univ.) or β-tubulin (Abcam) were used.