Agar 100 epoxy resin

For cryo‐preparation, the precipitated antibody was transferred into the 100 µm cavity of a 3 mm aluminum specimen carrier. This carrier was sandwiched with a flat 3 mm aluminum carrier and immediately high pressure frozen in an HPF Compact 01 (Engineering Office M. Wohlwend GmbH). The frozen samples were subsequently transferred into a Leica EM AFS‐2 freeze substitution unit (Leica Microsystems). Over a period of 4 days, samples were substituted in a medium of acetone containing 1% Osmium tetroxide. Freeze substitution was performed according to the following protocol: 30–40 h at –90°C; warm up at a rate of 2°C/h to –54°C; 8 h at –54°C; warm up at a rate of 5°C/h to –24°C; 15 h at –24°C; warm up at a rate of 5°C/h to 0°C; and 2 h at 0°C. At 0°C, samples were taken out and washed thrice in anhydrous acetone (on ice) and infiltrated with Agar 100 Epoxy resin (Agar Scientific), in a graded series of acetone and resin over a period of 3 days. Polymerization takes place at 60°C. Ultra‐thin sections with a nominal thickness of 70 nm were cut using a Leica UCT ultramicrotome (Leica Microsystem) and post‐stained with 2% aqueous uranyl acetate and Reynold's lead citrate. Examination regions on the sections were selected at random, examined with an FEI Morgagni 268D (FEI) operated at 80 kV. Digital images were acquired using an 11 megapixel Morada CCD camera (Olympus‐SIS). 

Transmission
electron microscopy (TEM) analysis was performed as previously described.^{19 (link)} Briefly, cells were fixed with 3% paraformaldehyde
and 2% glutaraldehyde in 0.1 M cacodylate buffer containing 5 mM CaCl₂ (pH 7.4) and then postfixed in 1% osmium tetroxide supplemented
with 0.5% potassium hexacyanoferrate trihydrate and potassium dichromate
in 0.1 M cacodylate for 1 h. The cells were then stained with 2% uranyl
acetate in water for 1 h, dehydrated in graded ethanol solutions,
and embedded in Agar 100 epoxy resin (Agar Scientific Ltd., Stansted,
UK). Ultrathin sections (70–90 nm) were viewed and photographed
with an FEI Tecnai SPIRIT (FEI, Eidhoven, Netherlands) transmission
electron microscope operated at 120 kV and equipped with a OneView
Gatan camera.

Transmission electron microscopy. Cells were fixed with 4% paraformaldehyde, 2% glutaraldehyde in 0.1 M cacodylate buffer containing 5 mM CaCl₂ (pH 7.4), then postfixed with 1% osmium tetroxide supplemented with 0.5% potassium hexacyanoferrate tryhidrate and potassium dichromate in 0.1 M cacodylate (1 h), stained with 2% uranyl acetate in water (1 h), dehydrated in graded ethanol solutions and embedded in Agar 100 epoxy resin (Agar Scientific Ltd., Stansted, UK). Ultrathin sections (70–90 nm) were viewed and photographed with a FEI Tecnai SPIRIT (FEI, Eindhoven, Netherlands) transmission electron microscope operated at 120 kV and equipped with a OneView Gatan Camera. To analyze ER-mitochondria contact sites by EM, we used the freehand drawing tool from FIJI software. We delimited only ER tubules within a 60 nm distance from the nearest mitochondrial surface, and delimited the mitochondrial perimeter. Then, we analyzed length and GAP of each contact site. We separated the contact site according to their GAP into three groups: from 0–15 nm, 15–30 nm, and 30–60 nm. Finally, we calculated the number of contacts per mitochondria, the average contact length, mitochondrial perimeter, total mitochondrial number, and ER-mitochondria contact coverage.

The shoot tips of spring shoot were washed with PBS (pH 7.2) at room temperature and post-fixed in 2% (w/v) OsO₄ in PBS (pH 7.2) for 3 h. The tissues were then rinsed twice in PBS and stained with uranyl acetate. The samples were dehydrated by passing them through an ethanol series and acetone, and they were then embedded in Agar100 epoxy resin (Agar Scientific). Thin sections were cut, treated with uranyl acetate/lead citrate, and examined with a Tecnai G2 Spirit transmission electron microscope (FEI; Phillips). Representative photographs are presented.

Hepatopancreas samples were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) and later rinsed in 0.1 M sodium cacodylate buffer prior to processing. Post-fixation was carried out in 1% osmium tetroxide/0.1 M sodium cacodylate buffer for 1 h. Tissues were washed in three changes of 0.01 M sodium cacodylate buffer and were subsequently dehydrated through a graded acetone series before embedding in Agar 100 epoxy resin (Agar Scientific, Agar 100 pre-mix kit medium). Embedded tissues were polymerised overnight at 60 °C. Semi-thin (1–2 µm) sections were cut and stained with Toluidine blue for viewing with a light microscope to identify suitable target areas. Ultra-thin sections (70–90 µM) of targeted areas were mounted on uncoated copper grids and stained with 2% aqueous uranyl acetate and Reynold’s lead citrate^{51 (link)}. Grids were examined using a JEOL JEM1400 transmission electron microscope and digital images captured using an AMT XR80 camera and AMT V602 software.