Veletta ccd digital camera

Cells were fixed with a mixture of 4% PFA and 0.05% glutaraldehyde (GA) for 10 min at room temperature, then washed with 4% PFA once to remove the residual GA and fixed again with 4% PFA for 30 min at room temperature. Next, cells were incubated with a blocking/permeabilizing mixture (0.5% BSA, 0.1% saponin, 50 mM NH₄Cl) for 30 min and subsequently with the primary monoclonal antibody against Lamp-1, diluted 1:500 in blocking/permeabilizing solution. The following day, cells were washed and incubated with the secondary antibody, the anti-mouse Fab fragment coupled to 1.4 nm gold particles (diluted 1:50 in blocking/permeabilizing solution) for 2 h at room temperature. Cells were then post-fixed as described in Polishchuk et al., 2019 (link). After dehydration, specimens were embedded in epoxy resin and polymerized at 60°C for 72 h. Thin 60 nm sections were cut on a Leica EM UC7 microtome. EM images were acquired from thin sections using a FEI Tecnai-12 electron microscope equipped with a VELETTA CCD digital camera (FEI, Eindhoven, the Netherlands). Morphometric analysis on the size of lysosome-like structures was performed using iTEM software (Olympus SYS, Germany).

For electron microscopy (EM), muscle tissues, fibroblasts, and myoblasts were fixed in 1% glutaraldehyde in 0.2 M HEPES buffer and post‐fixed in OsO4 and uranyl acetate. After dehydration through a graded series of ethanol, the samples were embedded in the Epoxy resin (Epon 812, Sigma‐Aldrich) and polymerized at 60°C for 72 h. Thin 60‐nm sections were obtained using a Leica EM UC7 microtome. EM images were acquired from thin sections using a FEI Tecnai‐12 electron microscope equipped with a VELETTA CCD digital camera (FEI, Eindhoven, The Netherlands). To count the number of mitochondria within the cells, EM images were acquired at the same magnification. Normal and abnormal mitochondria were manually counted on EM images.

The MDCK and HeLa cells transfected with UPIb-EGFP or UPIb/UPIIIa were fixed as described previously^{66 (link),67} . Cells were incubated with the anti-EGFP antibody (1:250; Abcam), washed in PBS and incubated in anti-rabbit Fab’ fragments coupled to 1.4-nm nanogold (1:100; Molecular Probes). Nanogold was enlarged by the GoldEnhance protocol (Nanoprobes Inc., Yaphank, NY, USA). Sections of 65-nm thickness were collected and EM images were acquired by a Techai^TM-G² Spirit electron microscope equipped with a VELETTA CCD digital camera (FEI, Eindhoven, The Netherlands).