Plan apochromat 63 1

For high‐speed live imaging, Xenopus embryos injected with ANKRD55‐GFP and mCherry‐CLUAP1 mRNA were anaesthetized with 0.005% benzocaine at stage 26. High‐speed in vivo imaging was acquired on a Nikon Eclipse Ti confocal microscope with a 63×/1.4 oil immersion objective at 0.267 s per frame. Kymographs were calculated using Fiji (Schindelin et al, 2012). Confocal images were collected with an LSM700 inverted confocal microscope (Carl Zeiss) with a Plan‐APOCHROMAT 63×/1.4 oil immersion objective. Bright field images were collected using a Zeiss Axio Zoom V16 stereo microscope with Carl Zeiss Axiocam HRc color microscope camera. Neural tube closure quantification was performed using Fiji. A two‐sample Kolmogorov–Smirnov test was used to compare distributions of control, morphant, and rescue embryos. Sample sizes were selected sufficient to determine moderate effects. Embryos were randomly selected from multiple clutches, and cells were randomly selected from individual embryos. No blinding to treatment was employed.

IFAs were performed on acetone-fixed parasites as described previously [7 (link)]. The following primary antibody dilutions were used: mouse anti-PvRALP1-Tr (1:50), rabbit anti-PvRON2 (1:100), rabbit anti-PvDBP (1:100), and rabbit anti-PvRhopH2 (1:100). The following secondary antibodies were used: Alexa Fluor 488 goat anti-mouse IgG (1:500; Invitrogen), Alexa Fluor 546 goat anti-rabbit IgG (1:500; Invitrogen) and 4′,6′-diamidino-2-phenylindole (DAPI) for nuclear staining (1:1,000; Invitrogen). The slides were mounted in ProLong Gold antifade reagent (Invitrogen) and visualized under oil immersion in a confocal scanning laser microscope (LSM710; Carl Zeiss MicroImaging, Thornwood, NY, USA) using a Plan-Apochromat 63×/1.4 oil differential interference contrast (DIC) objective lens. Images were captured with Zen software (Carl Zeiss MicroImaging) and processed with Adobe Photoshop (Adobe Systems, San Jose, CA, USA).

Fluorescence microscopy images were captured in vivo. For MpkC::GFP detection during germination, conidia were germinated for 2, 4, or 7 h at 37°C and observed using confocal microscopy. For stress treatments, 6 h germinated conidia were treated or not with 10 mM H₂O₂ for 10 min and observed within the next 10 min, or were germinated for 6 h in the presence of 1.2 M sorbitol. Image processing and fluorescence quantification were made using Image J and ZEN 2012 (Carl Zeiss, Jena, Germany). To observe conidiophores, the growing edge of a MpkC::GFP colony grown for 3 days at 37°C was sectioned, a drop of water was added and the section was carefully covered with a coverslip. Different fields in which conidiophore structure was better preserved were chosen for observation using confocal microscopy. To observe growing hyphae, 14 h grown mycelia was treated or not with 10 mM H₂O₂ for 20 min or grown for 14 h in MM containing 1.2 M sorbitol and then observed using confocal microscopy. All images were acquired using a Zeiss LSM800 inverted laser scanning confocal microscope using a Plan-Apochromat 63×/1.4 oil immersion objective and 488 and 561 nm laser lines. Maximum intensity projections were obtained from Z-stack images collected every 15 μm through entire cell volume. Images were processed using software ZEN 2012 (Carl Zeiss, Jena, Germany).

Confocal microscopy was performed using an LSM 800 microscope (Carl Zeiss) equipped with a Plan-Apochromat 63×/1.4 oil immersion objective.
During live cell microscopy pHluorin was excited at 488 nm and emitted light of 490–550 nm was detected. mRFP was excited by the 561 nm laser line and emitted light of 580–700 nm was detected. For fixed samples stained with AlexaFluor488 and AlexaFluor594 excitation was conducted at 488 nm and 561 nm and emitted light of 490–580 nm and 580–700 nm was detected.