Fv1000 d confocal microscope

Paraformaldehyde-fixed cells were permeabilized with 0.3% TritonX-100 in PBS and blocked with 3% FBS in PBST for 30 min. They were then incubated with anti-YAP antibody at 4 °C overnight and washed with PBS three times. Cells were incubated with anti-rabbit IgG-Alexa Fluor 488 for 1 h at room temperature, and washed with PBS three times. For confocal microscopy, cells were mounted in Prolong Gold reagent containing 10 μg/ml Hoechst 33342 (Life Technologies). When appropriate, cells were stained with phalloidin-Alexa Fluor 594 (Life Technologies) prior to mounting. Images were obtained with an FV1000-D confocal microscope equipped with a 40× objective lens using FV10-ASW software (Olympus). For screening of small molecules, PBS containing 10 μg/ml Hoechst 33342 was added and images were obtained by IN Cell Analyzer 2000 (GE Healthcare) using a 40× objective lens.

Cross-sections of the midportion of the gastrocnemius were cut at 10 µm in a cryostat (Microm Cryo-Star HM 560, Walldorf, Germany) and maintained at −20 °C until analyses. The sections were fixed in 0.1 M phosphate buffer containing 4% paraformaldehyde for 5 min and degreased in 100% methanol for 10 min at −20 °C. After being washed by phosphate-buffer saline (PBS), the sections were blocked in 10% donkey serum diluted with PBS containing 0.1% Triton-X 100 (PBS-T) for 20 min. Then, the sections were incubated overnight at 4 °C with anti-Laminin α2 (1:600; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Pax7 (1:100; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) and anti-MyoD (1:200; Santa Cruz Biotechnology) antibodies diluted in PBS-T containing 1% bovine serum albumin (BSA). Immunoreactivity was detected by incubation with Cy3-conjugated donkey anti-mouse IgG (1:500; Jackson ImmunoResearch, West Grove, PA, USA) and AlexaFluor 488-conjugated donkey anti-rabbit IgG (1:500; Life Technologies, Carlsbad, CA, USA) diluted in PBS-T containing 1% BSA for 4 h, Sections were counterstained with 4′,6-diamidino-2-phenylindole (Wako Pure Chemical Industries, Osaka, Japan). After several washes, the stained sections were mounted using the mounting medium (KPL, Gaithersburg, MD, USA). Images were acquired using an Olympus FV1000-D confocal microscope (Olympus, Tokyo, Japan).

Primary cultured hippocampal neurons were fixed in 4% paraformaldehyde for 1 h and permeabilized in 0.1% Triton‐X 100 for 10 min, followed by treatment with 1% bovine serum albumin for 20 min. These procedures were performed at room temperature (25°C). The following antibodies were used; anti‐IRE1 (1 : 500; Cell Signaling Technology, RRID: AB_823545), anti‐microtubule‐associated protein 2 (MAP2) (1 : 1000; EMD Millipore, Billerica, MA, USA, RRID: AB_11213363), anti‐MAP2 (1 : 500; Abcam, Cambridge, UK, RRID: AB_2138153), anti‐postsynaptic density protein 95 (PSD95) (1 : 500; NeuroMab, Davis, CA, USA, RRID: AB_2307331), anti‐XBP1s (1 : 500; BioLegend, Sandiego, CA, USA, RRID: AB_2562960), and Anti‐phospho‐IRE1α (1 : 500). Cells were visualized under a FV1000D confocal microscope (Olympus, Tokyo, Japan). P‐IRE1 and MAP2 fluorescence intensities 40–90 μm from somata in randomly selected dendrites were measured, using ImageJ (National Institutes of Health, Rockville, MD, USA). The number of P‐IRE1‐positive puncta overlapping with PSD95‐positive postsynaptic sites was manually counted 40–90 μm from somata in randomly selected dendrites. The cells were randomly chosen from five independent cultures.