Plapon60xosc na1

Murine embryonic fibroblast cells grown in a glass‐bottom dish were fixed with 4% PBS for 15 min at room temperature. After washing with 0.1% Tween 20 in PBS (PBST), cells were permeabilized with 0.5% Triton X‐100 in PBS for 5 min at room temperature and washed with PBST three times. The cells were then blocked with Blocking One solution (03953‐95; Nacalai Tesque) for 30 min at 4 °C, washed once with PBST, and incubated with a rabbit polyclonal anti‐p62 antibody (P0067; Sigma‐Aldrich) in Can Get Signal Immunostain Solution A (NKB‐501; Toyobo, Osaka, Japan) for 2 h, followed by extensive washes and incubation with Cy3‐conjugated donkey anti‐rabbit IgG antibody (AP182C; Life Technologies, Carlsbad, CA, USA) for 1 h. After washing with PBST three times, cells were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) and subjected to FM. For observation, an oil‐immersion objective lens (PLAPON60xOSC/NA1.40; Olympus) on the DeltaVision microscope system (GE Healthcare Life Sciences) was used as previously described 25.

Correlative light–electron microscopy was performed as previously described 26. Briefly, cells were fixed with 2.5% (w/v) glutaraldehyde for 1 h. Z‐stack optical images (typically 40–60 focal planes at 0.2‐μm intervals) were obtained using the Olympus oil‐immersion objective lens (PLAPON60xOSC/NA1.40) on the DeltaVision microscope system and subjected to deconvolution using standard software installed on the microscope system. After fluorescence imaging, samples were postfixed with 1% OsO₄ (3002; Nisshin EM, Tokyo, Japan), stained with 2% (w/v) uranyl acetate (8473–1M; Wako Pure Chemical Industries) for 1 h, dehydrated, and embedded in Epon812 (T024; TAAB Laboratory Equipment, Ltd., Reading, UK). Ultrathin sections (80 nm thick) were prepared using an ultramicrotome (Leica Microsystems, Wetzlar, Germany) and stained with 4% uranyl acetate, followed by a commercial ready‐to‐use solution of lead citrate (18‐0875‐2; Sigma‐Aldrich). Electron microscopy (EM) images were acquired using a JEM‐1400 electron microscope (80 kV; JEOL, Tokyo, Japan), and EM images were overlaid with the corresponding fluorescence images.