Ultracut uct ultramicrotome

Embedded samples were sectioned using an Ultracut UCT ultramicrotome equipped with a 35° diamond knife (Diatome) to acquire 32 serial sections with a mean thickness of 90 nm, which were stained as described in the Specimen preparation for conventional TEM section. TEM images were acquired of sections from the middle of the series to identify autophagosomal structures before relocation and imaging of the same structures in adjacent sections (between the fifth and 22nd sections). The TEM images were corrected for lens distortion using Distortion Correction plugin for FIJI (version 1.50g) and manually aligned into an image stack using GIMP (GNU Image Manipulation Program, version 2.8.16). The outermost membranes in images of each autophagosomal structure were manually segmented to using the TrakEM2 plugin for FIJI. The segmented data were interpolated (cubic interpolation; 8× interslice) and rendered as surfaces from two orthometric viewpoints using the Amira software package (FEI Technologies).

Hydrogels with and without TMV were embedded in epoxy resin (Spurr) after dehydration of the gels through a solvent series using different acetone : water mixtures (30%, 50%, 75%, 90%). The gels were incubated 20 min at each step. This was followed by staining with 2% uranyl acetate in acetone for one hour and two additional incubations in 100% acetone for 20 min. The samples were then immersed in a solution containing 33% Spurr and 67% acetone overnight without closing the vessels to allow the acetone to evaporate. Before the gels were put into embedding capsules and cured for at least 24 h at 60 °C, the solutions were twice exchanged with 100% Spurr for 1–2 h. The epoxide blocks were trimmed to the gel and ∼80 nm thin sections were prepared using a Leica Ultracut UCT ultramicrotome assembled with a diamond knife (Diatome, Nidau, Switzerland). The sections were placed on Pioloform covered 400 mesh copper grids and investigated using a Tecnai G2 Sphera FEI TEM (FEI, Eindhoven, Netherlands) at 200 kV. TEM images were recorded by a 16 megapixel camera (TemCam-F416 [4k × 4k], TVIPS, Gauting, Germany).

Postfixative was removed from cells and were rinsed with 0.1 M sodium cacodylate buffer pH 7.4 (5×1 minutes) on ice. Cells were washed with ddH2O at room temperature (5 × 1 minutes) followed by an ice‐cold graded dehydration ethanol series of 20%, 50%, 70%, 90%, 100% (anhydrous) for 1 minute each and 2×100% (anhydrous) at room temperature for 1 minute each. Cells were infiltrated with one part Durcupan ACM epoxy resin (44610, Sigma‐Aldrich) to one part anhydrous ethanol for 30 minutes, 3 times with 100% Durcupan resin for 1 hours each, a final change of Durcupan resin and immediately placed in a vacuum oven at 60°C for 48 hours to harden. Cells were identified, cut out by jewel saw and mounted on dummy blocks with Krazy glue. Coverslips were removed and 100 nm thick specimen sections were created with a Leica Ultracut UCT ultramicrotome and Diatome Ultra 45^°, 4 mm wet diamond knife. Sections were picked up with 50 mesh gilder copper grids (G50, Ted Pella, Inc) and carbon coated on both sides with a Cressington 208 Carbon Coater for 15 second at 3.4 volts.