Microphoto fxa epifluorescence microscope

Four- to 8-week-old female C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) were infected with 10³ or 10⁵ tachyzoites of the appropriate strains intraperitoneally. Mortality was observed daily for 30 days until the mice were sacrificed and the brains were harvested. The brains were homogenized with a Wheaton Potter-Elvehjem tissue grinder with a 100- to 150-μm clearance (ThermoFisher) in PBS, and an aliquot of the homogenate was viewed under a Microphoto-FXA epifluorescence microscope (Nikon) to look for GFP fluorescent cysts. Images of these cysts were analyzed with ImageJ to determine their sizes. ANOVA and Tukey HSD test were used to test for significance in cyst numbers and cyst sizes between strains in R. Survival statistics were analyzed by the R package survminer.

In order to examine the role of TfR1 during the infection of host cells by microsporidia, TRVb cells which do not express endogenous TfR and TRVb-1 cells which stably express the human TfR were utilized (these CHO cell lines were the kind gift of Drs. Parrish and McGraw, Cornell University) [64 (link)]. 1×10⁵ TRVb or TRVb-1 cells were cultured in six well plates and infected with 1×10⁶ spores. Fresh media was added every three days until the cells were fixed by 4% paraformaldehyde two weeks post-infection. Samples were washed with PBS followed by staining with 0.01% Calcofluor white (Sigma, USA) for 10 min at room temperature and the parasitophorous vacuole number (infection rate) of each well were counted using Microphoto-FXA epifluorescence microscope (Nikon).

At 1 day after the MEF cells were transfected with siRNA, fresh purified E. hellem spores were added to the MEF cell culture (1 × 10⁶ spores/well) to infect the MEFs. To quantify the E. hellem infection 2 days postinfection, the cultures were washed 3 times with PBS and then fixed with 4% paraformaldehyde. Spores in PVs were stained by the use of 0.01% Fluorescent Brightener 28 (Sigma-Aldrich catalog no. F3543) for 10 min at room temperature. Photomicrographs of 20 fields of each well were randomly taken at ×20 using a Microphoto-FXA epifluorescence microscope (Nikon), and quantification of the size and number of PV was performed using ImageJ.
To quantify the host mitochondrion association, infected MEF cells were stained with 100 nM MitoTracker mixed in incomplete DMEM for 30 min and then fixed with 4% paraformaldehyde. Cell nuclei were stained by the use of DAPI (1:1,000 dilution). Photomicrographs of 23 parasitophorous vacuoles from each group were randomly taken with a SP5 confocal microscope (Leica), and mitochondrion association was quantified by measuring the percentage of the PV membrane surrounded by host mitochondria using ImageJ.