Axioplan 2 light microscope

Following intravital imaging experiments or control ectopic implantation, constructs were recovered and fixed overnight in 2% paraformaldehyde at 4°C. Samples were decalcified in EDTA for 14 days at 4°C, embedded in NEG‐50 frozen section medium (Richard‐Allen Scientific, Thermo Fisher Scientific, Kalamazoo, MI, USA), and sectioned with a HM560 cryostat at 7 μm using SEC35 disposable steel blades (both from Thermo Fisher Scientific). Sections were either counterstained with Hoechst, stained with hematoxylin and eosin (H&E), or stained for tartrate‐resistant acid phosphatase (TRAP) visualizing osteoclasts, CD31 visualizing endothelial cells, or collagen type II using routine published protocols.28, 29, 30 CD31 staining was followed by H&E counterstain. Antibodies used for immunostaining are listed in Supporting Table 1. Images were taken on an Axioplan 2 light microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) using a Plan‐Neofluar 20 × /0.5 objective.

Adult insects from the infected colony were collected and dissected in 200 µl phosphate-buffered saline (PBS, pH 7.4) and the promastigote forms counted in a Neubauer chamber under a Zeiss Axioplan 2 light microscope (Oberkochen, Germany). Each intestinal compartment was macerated in PBS and the number of live promastigotes counted for each insect separately, according to the gut region.

Ten sunflower root pieces with O. cumana seedlings attached were cut from the sunflower plants of the rhizotron assay at 14, 21, 28, and 35 dpi using a binocular microscope. Samples were prepared as in Chabaud et al. (2022 ). Half of the samples were fixed in ethanol: acetic acid (3:1 by volume) for 10 min under vacuum, cleared in chloral hydrate 5 g/ml for 48 h under agitation and visualized with an Axioplan 2 light microscope (Zeiss, Jena, Germany). The remaining samples were fixed in FAA solution (10% formaldehyde, 5% acetic acid, and 50% ethanol) for 5 min under vacuum, dehydrated in alcohol series, and embedded in Technovit 7100 resin (Heraeus Kulzer, Germany). Thin sections of 10 µm were then made using a Reichert-Jung 2040 microtome (Leica Biosystems, Nussloch, Germany), stained with 0.2% toluidine blue for 3 min, mounted in DePeX mounting medium and scanned using a NanoZoomer image scanner (Hamamatsu Photonics, Japan). For the detection of phenolic compounds, hand-cut sections (with a razor blade) obtained from fresh root samples at 14, 21, 28, and 35 dpi were observed under epifluorescence (340–380 nm), as described in Lozano-Baena et al. (2007 (link)), in a Leica DM6 compound microscope with a Leica DFC7000 T digital camera (Wetzlar, Germany).

The force applied by the diamond knife during the cutting of serial EM sections compressed the sections. Section compression was calculated by dividing the width of the tissue block face in the resin with the width of EM sections measured perpendicular to the diamond knife blade (X–Y plane). The measurements were performed by Zeiss Axioplan2 light microscope. There was no change in the EM section dimension in the direction parallel (X plane) with the diamond knife edge.

Seventy-two hpf control and bbs5 ATG MO injected (mild to moderate phenotype) embryos were euthanised in 4% tricaine and fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer for two hours at room temperature. They were then dehydrated in a stepwise manner in acetone then impregnated with epoxy resin in stepwise fashion. Embryos in 100% epoxy resin were then placed individually in coffin moulds (EMS #70905-01) and polymerised at 60°C for 24 hours. One micrometer transverse microtome sections were stained with 1% toluidine blue in 1% borax. In parallel, zebrafish were also embedded in (Electron Microscopy Sciences, Hatfield, PA, USA) and allowed to set in a low humidity environment. A glass knife was used to cut 5 μm sections which were then stained by Lee’s trichrome and mounted in Histamount. Sections were then analysed on a Zeiss Axioplan 2 light microscope and images were taken (Zeiss AxioCam HRc camera).

The head regions with tentacles were fixed overnight at 4 °C in a 2.5% solution of glutaraldehyde in 0.2 M phosphate buffer (PBS). The heads were then washed in 0.2 M PBS for 4 h with three changes and postfixed in 1% OsO₄ in 0.2 M PBS for 3 h at room temperature (RT) with gentle rotation. The specimens were then dehydrated in an increasing series of ethanol concentrations (from 15 to 96%) and isopropanol. They were subsequently infiltrated in a mixture of isopropanol and Spurr resin for 3 days and then embedded in pure Spurr resin at 60 °C for 24 h.
The anterior part of the body of two adults embedded in resin were used to prepare a complete series of 1-μm (semi-thin) and 70-nm (thin) resin sections with a Leica UC 7 ultramicrotome (Leica Microsystems, Wetzlar, Germany). The semi-thin sections were stained with methylene blue and examined with a Zeiss Axioplan2 light microscope equipped with an AxioCam HRm camera (Carl Zeiss Microscopy, LLC, USA). Semi-thin sections were used for description of gross anatomy and for 3D reconstructions. The thin sections were stained with uranyl acetate and lead citrate and were examined with a JEM-1011 JEOL or a JEM-100 B-1 JEOL transmission electron microscope (JEOL, Akishima, Japan).