Low melting point agarose

Fluorescence microscopy was performed as described (Takaki et al., 2013; Yang et al., 2012 ). Quantification of bacterial burdens, assessments of mycobacterial cording, and enumeration of neutrophils were performed with a Nikon Eclipse Ti-E inverted microscope fitted with 2×, 10×, and 20× objectives. Enumeration of macrophages, assessments of intracellular bacterial growth, measurements of granuloma diameter, and evaluation of histological sections were performed on a Nikon Eclipse E600 upright microscope fitted with 10× and 20× objectives. For laser scanning confocal microscopy, larvae were anesthetized in N-phenylthiourea (PTU)-supplemented fish water containing 0.025% Tricaine and embedded in 1.5% low-melting-point agarose on optical bottom plates or dishes (MatTek Corporation). A Nikon A1 confocal microscope with a 20× Plan Apo 0.75 NA objective was used to generate 40 μm z stacks consisting of 1.3–2 μm optical sections. The galvano scanner was used for all static imaging and for time-lapse imaging of the CHT. Time-lapse images were taken at 5 min intervals for 8 hr. Data were acquired with NIS Elements (Nikon). Macrophage tracks were generated using the 3D tracking feature of Imaris (Bitplane Scientific Software).

Fluorescence microscopy was performed as described (Takaki et al., 2013 (link)). Quantification of camptothecin-induced apoptosis by acridine orange staining and assessments of mycobacterial cording were performed with a Nikon Eclipse Ti-E inverted microscope fitted with 4x and 10x objectives. For laser scanning confocal microscopy, anesthetized larvae were embedded in 1.5% low-melting-point agarose on optical bottom plates or dishes (MatTek Corporation). For long term microscopy, agarose was covered with fish water containing 0.007% Tricaine. A Nikon A1R confocal microscope with a 203 Plan Apo 0.75 NA objective was used to generate 35-40 μm z stacks consisting of 0.3-2 μm optical sections. The galvano scanner was used for all static imaging and for time-lapse imaging of the CHT. Time-lapse images were taken at different intervals for up to 5 hr. Data were acquired with NIS Elements (Nikon). A heating chamber (Okolab) adapted to the microscope was used to maintain temperature constantly at 28.5°C during the imaging process. In Figure 1B, a single z image is shown for the control panels to appreciate cellularity and maximum intensity projections for TNF-high panels to appreciate cording. Confocal images are pseudocolored to facilitate visualization.