Ec plan neofluar 10x 0.3 objective

Cells were grown to ~1×10⁶ cells/ml and loaded into polyglutamate-coated motility chambers^{33 (link)} sealed with Vaseline. Thirty second videos were acquired under dark-field illumination at ~40 frames per second (fps) using a Hamamatsu ORCA-Flash 4.0 camera on a Zeiss Axio Imager A2 microscope with an EC Plan-Neofluar 10x/0.3 objective lens and Zen 2.6 Pro software. At least five videos were collected for each independent biological replicate. Single cell tracking and mean squared displacement were performed in MATLAB as previously described for fluorescent parasites^{33 (link)}. For “high density” motility analyses, cells were grown to ~1×10⁷ cell/ml, then diluted in conditioned medium to ~1×10⁶ cells/ml and analyzed as above.

Mineral deposition was evaluated by intraperitoneal injections of calcein (Sigma # C0875; 2.5 mg/kg body weight) and alizarin complexone (Sigma # a3882; 7.5 mg/kg) into pregnant females and, postnatally, into cubs. Two mice were used for each injection regime. Prenatally harvested limbs were fixated overnight in 4% PFA/PBS, dehydrated to 100% ethanol, embedded in paraffin and sectioned at a thickness of 7 μm. Postnatally harvested limbs were fixated 24 h in 4% PFA/PBS and gradually dehydrated from 70% ethanol to 100% ethanol twice for 48 h each time. Then, samples were infiltrated and embedded in JB-4 Embedding Kit (Electron Microscopy Science #14270–00) and sectioned longitudinally at a thickness of 7 μm. Fluorescence was visualized by confocal microscopy.
Confocal imaging was performed using a Zeiss LSM 510 upright confocal microscope (Carl Zeiss, Jena, Germany) with an EC Plan-Neofluar 10x/0.3 objective, NA 1.0. Calcein fluorochrome was excited with a 488 nm argon laser and alizarin with 561 nm argon laser. Following imaging, all images of the same section were stitched using Microsoft Image Composite Editor (version 1.4.4.0). Contrast was increased by using the “auto contrast” tool of Google Picasa (version 3.9.137) and Matlab’s “imadjust” function.

C57BL/6 mice were inoculated i.v with 150,000 WT PbANKA or IL-6 Tg-PbANKA/LISP2 SPZ and livers were harvested at 24 h and 40 h post-infection. EEF density and dimensions were determined by microscopy on freshly dissected livers using a spinning-disk confocal system (UltraView ERS, Perkin Elmer) controlled by Volocity (Perkin Elmer) and composed of 4 Diode Pumped Solid State Lasers (excitation wavelengths: 405 nm, 488 nm, 561 nm and 640 nm), a Yokogawa Confocal Scanner Unit CSU22, a Z-axis piezoelectric actuator and a Hamamatsu Orca-Flash 4.0 camera mounted on a Axiovert 200 microscope (Zeiss). Z-stacks of images spaced 3 µm apart and covering 18 to 60 µm were acquired using a EC Plan-NEOFLUAR 10x/0.3 objective (Zeiss) or a Plan-APOCHROMAT 63x/1.4 oil objective (Zeiss). Image analysis was performed with the Fiji software (14 (link)). Z-stacks were converted to maximum intensity projections and the resulting images were segmented using the pre-implemented Triangle algorithm. EEFs were manually selected with the Magic Wand tool to determine their dimensions (area, Feret’s diameter).