150 μm grids

Detailed CLEM method was recently published^{59 (link)}. Briefly, p62-GFP^KI/+ MEFs were cultured on coverslips etched with 150-μm grids (Matsunami Glass Ind., Ltd.). The MEFs were treated by sodium arsenite (As[III]) for 10 hr and then cultured in regular medium for 3 h. They were fixed with 2% PA–0.5% glutaraldehyde (GA) in 0.1 M PB (pH 7.4), and phase-contrast and fluorescence images were obtained using a confocal microscope (FV1000; Olympus). The cells were fixed again with 2% PA and 2% GA in 0.1 M PB (pH 7.4), processed using the reduced-osmium method, and embedded in Epon812. Areas containing cells of interest were trimmed, cut as serial 70-nm or 200-nm sections, and observed with an electron microscope (EM; JEM1400 Plus; JEOL). Alignment of light microscopy and EM images was performed using TrakEM2 software version 1.0a 2012 -07-04^{60 (link)} (https://imagej.net/TrakEM2).

Cells were cultured on coverslips coated with 150 μm grids (Matsunami Glass Ind.). The cells were stimulated with DMXAA (25 µg ml⁻¹) in the presence of protease inhibitors (E64d (30 µg ml⁻¹) and pepstatin A (40 µg ml⁻¹)) and orlistat (20 µg ml⁻¹). Cells were fixed with 2% PFA–2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 15 min at room temperature and rinsed three times for 15 min each time in 0.1 M phosphate buffer (pH 7.4). The fluorescence images were obtained using a confocal microscope (LSM880 with Airyscan (Zeiss)). They were fixed again with 2% PFA–2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for more than 15 min at 4 °C, and then with a reduced osmium fixative. After embedding in Epon812 resin, areas containing cells of interest were trimmed according to the light-microscopic observations, and serial ultrathin sections (80 nm thickness) were prepared and observed with an electron microscope (JEM1400EX; JEOL)^{50 (link),51 (link)}.