Zen 3.6 blue edition

Trypanosomes were harvested by centrifugation and fixed with 4% paraformaldehyde in Phosphate Buffered Saline (PBS, supplemented with 250 mM sucrose in case of RNAi experiments) for 15 min at 4°C. After two washes, the fixed cells were resuspended in PBS and immobilized on poly-L-lysine (Sigma) coated wells, further permeabilized with PBS containing 1% Triton X-100 and blocked with blocking buffer (PBS containing 3% BSA and 0.25% Tween 20). To study the subcellular localization, α-TbAldolase (1:500 dilution in blocking buffer) was used as glycosomal marker, while Rabbit Alexa Fluor 594 (Thermo Fischer Scientific) at 1:1000 in blocking buffer was used as secondary antibody. The Nuclear and kinetoplast DNA were stained with DAPI. Stained cells were layered with Mowiol (Sigma) antifade-medium and covered with coverslips. After an overnight setting time that allows the polymerization of Mowiol, the images were captured using Zeiss ELYRA Super Resolution Microscopy and analyzed using Zen 3.6 (blue edition) (Carl Zeiss Microscopy GmbH).

Fixed preparations were imaged with a Zeiss LSM 900 or 800 laser scanning confocal equipped with an Axio Imager.Z2 microscope. A 10×/0.3 EC Plan-Neofluar M27 or 40×/1.40 NA Oil Plan-Apochromat DIC M27 objective lens was used. The software program used was Zen 3.6 (blue edition) (Zeiss AG).

Whole mount in-situ hybridization was performed as previously described [36 (link),37 (link)], using riboprobes for E-cadherin and Dermo-1. Permeabilization of the tissue was performed with proteinase K (10 μg/mL) for 10–40 min at room temperature, depending on the size of the embryo. Afterward, 1 μg/μL probe was hybridized for 48 h at 65 °C. The hybridized probe was visualized by an anti-DIG antibody conjugated to alkaline phosphatase (Sigma Aldrich, St. Louis, MO, USA).
Following ISH, the embryos were photographed with a stereo microscope (M165 FC, Leica, Wetzlar, Germany) equipped with a digital camera (DFC420 C, Leica, Wetzlar, Germany). The resulting photos were assembled in figures by using InkScape software (Open source software, Version 1.2.1, 2022).
For vibratome sectioning after in-situ hybridization, the embryos were embedded in 2.5 − 4% agarose gel and sectioned with a vibratome (VT 1000 S, Leica, Wetzlar, Germany) to 50μm. Sections were collected and covered with cover slips and Aquatex (Merck, Darmstadt, Germany). The sections were scanned using the AxioScan (Zeiss, Aalen, Germany) and edited with the microscopy software ZEN 3.6 (blue edition) (Zeiss, Aalen, Germany).