Axiovert observer z1

Tissue sections were examined and images captured using a Zeiss 510 laser scanning confocal microscope with UV (405 nm), argon (488 nm), helium/neon (546 nm), and helium/neon (633 nm) lasers. All images were acquired as 10–30 μm Z-stacks (in 1–2 μm intervals) and analyzed as projections using the LSM 510 Image Browser software (Zeiss, Oberkochen, Germany). Epifluorescent and light microscopy images were acquired with a Zeiss Axiovert Observer Z1 epifluorescent/light microscope equipped with an AxioCam cooled-color camera (Zeiss). Images were exported to Adobe Photoshop CS5 (Adobe Systems Inc., San Jose, CA, United States) and further processed for contrast when necessary.

Parasite ingestion was determined using modifications of a previously reported assay (16 (link)). In brief, mCherry was expressed in inducible Chinese hamster ovary (iCHO) cells with the addition of 2 μg/ml of doxycycline (DOX). Parasites from infected iCHO cells were harvested, purified, treated with pronase and saponin, and imaged on Cell-Tak (Fisher Scientific)-coated slides using a Zeiss Axiovert Observer Z1 inverted fluorescence microscope. For each biological replicate, more than 97 parasites of each genotype or treatment were enumerated for host-derived mCherry accumulation within parasites. Samples were coded during the time of harvesting to blind the experimenter during imaging and quantification.
In this study, we refer to the in vitro conditions that promote tachyzoite conversion to bradyzoite cysts as “bradyzoite-inducing conditions.” This includes the use of alkaline (conversion) media and growth without CO₂. For all experiments, 2 μg/ml DOX was used. Detailed changes for each ingestion assay are described below.

All phase-contrast and fluorescence imaging was performed on a Zeiss Axiovert Observer Z1 inverted fluorescence microscope. Exposure times within a given experiment were kept constant for all samples.

Live microscopy was performed as previously described [16 (link)] using a Zeiss Axiovert Observer Z1. Sf9 cells infected with AcMNPV1-ANCHOR3 were observed using a ×63 objective. Image analysis was performed using Fiji software. Visualization of the S. exigua larva infected by recombinant AcMNPV was carried out using a ×10 objective.
Fluorescence quantification was performed using 96-well, black flat-bottom plates filled with 100 µL of supernatant of infected cell culture at various times post-infection, using as control supernatant of wildtype (Wt) AcMNPV infected cells. In parallel, each sample titer was determined by plaque assay as indicated previously. The excitation wavelength used was 485 nm and the emission wavelength was 530 nm (bandwidth 20 nm for both). The intensity of fluorescence was measured in triplicate on a fluorescence reader Tecan Spark™ 10M microplate reader using SparkControl V1.2.20 software.

Encapsulated cell viability was assessed on days 1, 7 and 21 by calcein acetoxymethyl ester/ethidium homodimer fluorescent stain as described previously.1 (link) Briefly, hydrogels were submerged in media, 2 mM calcein acetoxymethyl ester, and 4 mM ethidium homodimer (LIVE/DEAD^® Viability/Cytotoxicity Kit for mammalian cells; Life Technologies, Grand Island, NY). Viable cells within the hydrogel sheets were identified based on intracellular enzymatic conversion of non-fluorescent calcein-AM to green fluorescent calcein while ethidium homodimer-1 penetrated damaged membranes of dead cells and upon binding to nucleic acids fluoresced bright red. After 10 min in a tissue culture incubator, each hydrogel was imaged under an epifluorescent microscope (Zeiss Axiovert Observer Z1) and 10–15 optical slices at 28 µm intervals were taken in the z-plane at three diagonal positions (e.g., top left, center, and bottom right) in the x–y coordinates. Green fluorescent images for live cells and red fluorescent images for dead cells for each optical slice were separately processed on ImageJ software (Rasband, National Institutes of Health) to obtain cell counts.