Zebrafish at the prim25 stage (at approximately 36 hours post fertilization; staged according to the method of Kimmel, et al. (Kimmel et al., 1995 (link))), were manually dechorionated, and anesthetized in Tris-buffered tricaine methane sulfonate (Tricaine; 200 mg/ml; Western Chemicals, Inc., Frendale, WA) prior to injection. For injections, approximately 5 nL of opsonized polystyrene bead suspension at 1.5 × 107 beads/ml in PVP, or 2 nL of C. albicans suspension at 1.0 × 107 cfu/ml was microinjected through the otic vesicle into the hindbrain ventricle to achieve a dose of approximately 15 beads or 15 fungal cells at the site of injection. Within one hour post-injection, larvae were screened using a Zeiss Axiobserver Z1 microscope equipped with Vivatome system (Carl Zeiss Microimaging, Thornwood, NJ) for selection of larvae containing 13–17 beads/fungal cells at the site of injection (SOI). Larvae were subsequently incubated at 33°C in E3 media containing PTU. In experiments to test the relationship between extracellular fungal number and mortality, larvae were preincubated for 1 hour pre-infection with either DMSO or diphenyleneiodonium (Enzo, Farmingdale NY) as described (Brothers et al., 2013 ).
Vivatome system
Manufactured by Zeiss
The Vivatome system is a microscope imaging equipment developed by Zeiss. It is designed to capture high-quality images of living samples, enabling researchers to observe and study biological processes in their natural state.
Automatically generated - may contain errors
2 protocols using vivatome system
1
Zebrafish Hindbrain Infection Assay
2
Mitochondrial Morphology Analysis Protocol
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Cells were seeded into Nunc Lab-Tek chamber slide at 5000 cells per chamber. Briefly, cells were fixed with 4% paraformaldehyde then permeabilized with 0.15% Triton X100 before 45 min of 10% bovine serum albumine saturation. Overnight incubation of primary antibodies anti-Tom20 (abCam) was performed before fluorescent-secondary antibody incubation. Cells were finally rinsed twice with PBS. Fresh PBS was added to the chambers to maintain cells integrity. Fluorescence microscopy was performed on a Zeiss Axio Observer microscope equipped with the Vivatome system with a 63× oil spring-loaded objective. The images were acquired using AxioVision (6D acquisition and vivatome modules). A minimum of 30 different cells obtained from three different experiments was randomly selected per experimental condition, and the analysis of mitochondrial morphology was performed using Morphostryder (Explora Nova).
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