For serial block-face SEM imaging, whole retinas were fixed in a mixture of 2.5% glutaraldehyde and 2% formaldehyde in sodium cacodylate buffer with 2 mM calcium chloride at RT for 5 min, and then on ice for an additional 2–3 h. Fixed retinas were stained with osmium tetroxide and uranyl acetate, and mounted and stained with lead aspartate for serial 3-D SEM as described previously (Pfeifer et al., 2015 (link)). The trimmed, resin-embedded stained blocks were imaged using a 3View serial block-face imaging system (Gatan) installed on a SIGMA-VP (variable pressure; Carl Zeiss) SEM, operating at an accelerating voltage of 1.5 kV using a standard 30-µm condenser aperture. The SEM was operated in high vacuum. Data were collected with a pixel size of 5.75 nm in the x–y plane and 25 nm along the z axis. Individual block-face electron micrographs were locally aligned with IMOD software (Kremer et al., 1996 (link)). The synaptic terminals of bipolar cells were recognized in the inner plexiform layer (IPL) by the presence of synaptic ribbons, vesicles, mitochondria, and their distinct morphology. Full 3-D reconstructions of the two bipolar terminals were made by two blind users by hand segmentation of the plasma membrane, ribbons, mitochondria, and microtubule structures of the individual serial images in Amira software (FEI) and TrakEM2 software (FIJI).