Cm 10 tem

For histological analysis, 3μm thick sections were stained with basic fuchsine to evaluate the presence of intracellular glycolipid as dark blue material in contrast with reddish cytoplasm and nucleus. As indicated by the present guidelines, “disorders of the lysosomal type require electron microscopy for morphological diagnosis on tissue biopsy” (15 (link)). For this reason, samples were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer, at pH 7.3, post-fixed in osmium tetroxide 1,33% in the same buffer and processed following a standard schedule for embedding in Epon resin. Ultrathin sections were obtained by a diamond knife and stained with uranyl acetate and lead hydroxide. A Philips CM-10 TEM or Jeol 1400-plus TEM were used for observation and photographic analysis.

unc-42(e270) was fixed as previously described (White et al., 1986 (link)). These fixed worms were then cut into 50 nm sections using RMC Powertome XL and collected onto grids. The nerve ring region of unc-42(e270) was then imaged either manually with a Phillips CM10 TEM or automatically with a JEOL 1400Plus TEM and the SerialEM software. Sections were then aligned and montaged, all of the axons in the nerve ring were serially traced, and synapses were annotated using the TrakEM2 software (Cardona et al., 2012 (link)). The region imaged, reconstructed, and annotated was ~15 μm in length and included 309 serial sections. Neurons were identified by characteristic synaptic and/or morphological features together with relative cell body position (Supplementary file 6). Synapse counts and axon adjacency counts were then extracted using scripts kindly provided by Christopher Brittin (Brittin et al., 2018 (link)). To compare to the unc-42(e270) synapse and axon adjacency counts to the previously described wild type (N2U) (Cook et al., 2019 (link)), the sections were aligned from the beginning of the RMEV neuron nucleus, a neuron that is easily identifiable based on morphology and position, to the anterior end of the nerve ring.