Eclipse ni microscope

Synchronous ring-stage SUB2HA3:loxP parasites were mock -or RAP-treated, then ~44 hr later schizonts were Percoll-enriched and added to fresh RBCs for 4 hr to allow egress and invasion. Without an intervening centrifugation step, a sample of the culture was supplemented with Hoechst 33342 (2 μg/ml; ThermoFisher) and Alexafluor 488 Phalloidin (diluted 1:50; ThermoFisher) and immediately applied to a viewing chamber for live imaging as described previously (Collins et al., 2013b (link)). Z-stack images were acquired at 2 μm intervals using a Nikon Eclipse Ni microscope with a 100x Plan Apo λ HA 1.45 objective and a Hamamatsu C11440 digital camera. The number of phalloidin-labelled ghosts per image was counted manually for each treatment.

MZybx1 and wild-type control embryos were collected at the same time and grown at 28.5°C until the 75% epiboly stage. They were subjected to temperature shifts to 22°C until the 21-som stage and shifted back to 28°C until 5 dpf. At 24 hpf, embryos were treated with the phenylthiourea at 0.003% concentration in Danieau's solution to prevent pigment formation. To distinguish between general LR and heart morphogenesis defects, embryos were sorted for heart looping and only embryos with normal looping were assessed for cardiac valve function. Prior to imaging on day 5, embryos were immobilised by adding 25× tricaine solution to Danieau's solution and mounted in 0.6% low melt agarose on a 3-cm glass cover slip-bottom Petri dish for imaging at 13 frames per second using Nikon ECLIPSE Ni microscope with a HAMAMATSU digital camera C11440, ORCA-Flash4.OLT. Time-lapse movies of approximately 30 s to 1 min duration were acquired.
To analyse the direction and velocity of the blood flow in ybx1 and control embryos, the particle image velocimetry (PIV) application was used in MATLAB (Thielicke and Sonntag, 2021 (link)). Each movie was converted in ImageJ using the function ‘find edges’ and uploaded to PIV, the region of interest was set out to be a junction in the AV canal and frames were analysed. Vectors were saved and analysed for direction with a custom MATLAB code.

Thin films of parasite cultures were made on glass slides, then air dried and stored under dessicant at −80°C. Slides were thawed, fixed in 4% paraformaldehyde for 30 min then permeabilized in 0.1% Triton X-100 in phosphate buffered saline (PBS) for 10 min prior to blocking in 3% (w/v) BSA in PBS overnight. Antibody incubations were carried out for 30 min at 37°C in a humidified chamber followed by washing twice for 5 min each in PBS. All antibodies were diluted into 3% (w/v) BSA in PBS. For microscopic imaging, samples were mounted in Vectashield containing DAPI (Vector laboratories). Images were acquired using a Nikon Eclipse Ni microscope with a 100x Plan Apo λ HA 1.45 objective, with a Hamamatsu C11440 digital camera and processed using Fiji.