Uc6 microtome

Flat embedded samples were mapped using binocular to map the precise region of interest at the basal part of the ventricle. For the ultramicrotome sectioning, samples were carefully oriented inside the holder according to the correlation maps derived from the embedded samples. Polymerized flat blocks were trimmed using a 90° diamond trim tool. The arrays sequential sections of 100 nm were generated using a 35° ATC diamond knife (Diatome, Biel, Switzerland) mounted on Leica UC6 microtome (Leica, Vienna). Arrays that covered the span of the ROI were transferred on pieces of silicon wafer [20 (link)]. Wafers were analyzed using an FEI Helios Nanolab 650 scanning electron microscope (Thermo Fischer, Eindhoven). Wafers were screened to target the relevant sections, and the ventricle area was imaged by tiling multiple images using the following imaging settings: MD detector, accelerating voltage 2 kV, current 0.8 nA, and dwell time 4–6 μs. Subsequently, relevant areas were re-imaged using higher resolution parameters, collected either manually or using the AT module of MAPs program (Thermo Fischer, Eindhoven) [20 (link)]. For electron microscopy data interpretation, previous reports in the literature were used to recognize the different neural cell types based on their ultrastructural characteristics [5 (link),21 (link)].

Each specimen was prepared as described previously (Lee et al., 2010 (link)). To prepare semi-thin sections, shoot tips were fixed in a solution containing 2.5% (v/v) glutaraldehyde in 0.1 M PBS buffer (pH 7.2) under vacuum conditions for 30 min. After fixing for 2 days at 4°C, the shoot tips were rinsed twice with phosphate buffer and then dehydrated at room temperature in a graded series of ethanol starting at 30% then going to 40%, 50%, 60%, 70%, 80%, 90%, 95%, and 100% (v/v). The specimens were embedded in Spurr’s low-viscosity embedding medium. Infiltration was started in a 2:1 ethanol/embedding medium. The specimens were left in the mixture with swirling of the container for 2 h after which the medium was replaced with a 1:1 ethanol/embedding medium and left in the container, which was swirled, for 4 h. Next, the medium was replaced with 1:3 ethanol/embedding medium and incubated with swirling of the container for another 6 h. After that, the mixture was drained and embedding medium was added to the container and left overnight. After curing the specimens for 48 h at 65°C, the specimens were mounted and sectioned at a thickness of 1 μm using a UC6 microtome (Leica, Wetzlar, Germany). After staining briefly with 0.1% toluidine blue, the sections were observed under bright-field optics using an Axio Imager M2 microscope (Zeiss).