Uc6 ultramicrotome

Wild-type Drosophila first instar larval central nervous systems were manually dissected by mechanical separation of the anterior tip of the larva from the posterior portion in PBS, and immediately transferred to 2% glutaraldehyde in 0.1 M Na-cacodylate, pH 7.4 buffer. Samples were post-fixed in 1% OsO₄ in the same buffer and stained en bloc with 1% aqueous uranyl acetate before subsequent dehydration in ethanol and propylene oxide, and embedding in Epon. Serial 45 nm sections were cut with a Leica UC6 ultramicrotome using a Diatome diamond knife, and picked up on Synaptek slot grids with Pioloform support films. Sections were stained with uranyl acetate followed by Sato’s lead (Sato, 1968 (link)). Sections were then imaged at 4.4 nm × 4.4 nm resolution using Leginon (Suloway et al., 2005 (link)) to drive an FEI Tecnai 20 transmission electron microscope. The resulting 77,000 image tiles were contrast-corrected, montaged and registered with TrakEM2 (Cardona et al., 2012 (link)) using the nonlinear elastic method (Saalfeld et al., 2012 (link)). The generated data volume of 22,775×18,326×462 voxels corresponds to a volume of 91×73×21 µm³. The data set covers approximately the posterior half of abdominal segment A2, and a nearly complete abdominal segment A3.

HeLa cells were fixed in 3% formaldehyde with 0.1% glutaraldehyde, dehydrated in an ethanol series and embedded in LR White resin. Ultrathin sections prepared using a Leica UC6 ultramicrotome and Diatome 45° diamond knife were immunolabelled and contrasted according to Stradalova et al. [33 (link)] using mouse anti-PI(4)P antibody (Echelon Biosciences, Z-P004, 20 μg/mL) and goat anti-mouse IgM conjugated to 6 nm gold nanoparticles (Jackson ImmunoResearch, 115-195-075, dilution 1:30). For double staining (Figure 4), ultrathin sections were incubated with purified OSH1-PH domain tagged with GST, washed and then immunolabelled using anti-PI(4,5)P2 antibody (Echelon Biosciences, Z-A045, 16 μg/mL), rabbit polyclonal anti-GST antibody (a gift from Igor Shevelev, 5 μg/mL) and the secondary antibodies goat anti-rabbit IgG conjugated to 6 nm gold nanoparticles (Jackson ImmunoResearch, 111-195-144, dilution 1:30) and goat anti-mouse IgM conjugated to 12 nm gold nanoparticles (Jackson ImmunoResearch, 115-205-075, dilution 1:30).
Images were acquired using a Jeol JEM 1400 Flash, operated at 120 KV, equipped with a Hamamatsu Orca Flash CMOS camera.
Clustering and co-localisation analyses were performed using our self-developed Gold plugin [34 (link)] for the Ellipse software version 2.0.8.1 (ViDiTo, Slovakia).