Dolichos biflorus agglutinin dba

The following antibodies and dilutions were used on paraffin-embedded sections for immunofluorescence staining: anti-PS (Regulus Therapeutics Inc, 1:1,000), anti-Pparα (Abcam ab8934, 1:200), anti-Pmp-70 (EMD Millipore ABT12, 1:200), anti-phosphohistone H3 (1:400, Sigma-Aldrich H0412). Secondary antibodies were conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes, 1:400). Lectins used were Dolichos biflorus agglutinin (DBA; Vector Laboratories, 1:400) and Lotus tetragonolobus agglutinin (LTA; Vector Laboratories, 1:400). Tissue sections were stained as described43 (link). TUNEL assay was performed using the Promega Dead End Tunel Fluorometric System Kit per the manufacturer's directions with the following modification: the proteinase K treatment time was extended to 15 min. Quantification of apoptosis and proliferation was performed by randomly selecting 10 × 20 magnification fields per sample and subsequently determining the percentage of positively stained cyst epithelial cells. The person performing the quantification was blinded to the genotype of kidney sections.

ME49 parasites were grown in HFFs and standard medium, before changing to alkaline-stress medium at 24 h post-infection. At this time point, to remove residual standard medium, the infected HFF monolayer was washed once with alkaline-stress medium. Parasites were grown for an additional 48 h at 37°C at ambient CO₂ to allow for stage conversion. The medium was then aspirated and HFF monolayers rinsed with PBS. Following addition of 3 ml PBS, the host-cell monolayer was detached by scraping, followed by the mechanical release of parasites by serially passing once through 27- and twice through 30-gauge needles before filtering through a polycarbonate filter with a 3-µm pore size (Whatman). The efficiency of stage conversion was estimated at 98%, as assessed by immunofluorescence staining and microscopy at the time of the Bz harvest. Guinea-pig anti-CDPK1, diluted 1:1000, provided a general parasite stain, while fluorescein-labeled dolichos biflorus agglutinin (DBA; Vector Laboratories), diluted 1:150, served as an early-Bz marker. DBA is a lectin that recognizes N-acetylgalactosamine on the Bz-specific cyst-wall protein CST1 (Tomita et al., 2013 (link)).

Tachyzoite-to-bradyzoite differentiation using in vitro alkaline switch was performed as described by Knoll and colleagues (110 (link)). The differentiation medium consists of RPMI 1640 without bicarbonate and supplemented with 2.05 mM l-glutamine (HyClone), 20 mM HEPES free acid (IBI Scientific), 1% Glutamine XL (a stable form of glutamine; VWR), 1% FBS, and 1% penicillin-streptomycin. The differentiation medium pH was adjusted to 8.1 with sodium hydroxide. Monolayers of HFF cells were cultured on circular glass coverslips until confluent and infected with type II parasite strains at an MOI of ∼0.5. Infected cells were washed once 3 h later with DPBS supplemented with Ca²⁺ and Mg²⁺, and cultures were returned to high-pH differentiation medium for 3 days at 37°C in ambient air to differentiate cysts. Infected cells were fixed in 4% paraformaldehyde, and the excess of paraformaldehyde was quenched with 0.1 M glycine. Infected cells were simultaneously permeabilized and blocked for 30 min at room temperature in 3% FBS–0.2% Triton X-100 and incubated for 1 h at room temperature with a 1:250 dilution of rhodamine-labeled Dolichos biflorus agglutinin (DBA) (Vector Laboratories). Preparations were washed three times with DPBS, mounted in SlowFade gold antifade with DAPI (Life Technologies), and imaged with a Nikon A1R SI confocal microscope (Nikon, Inc.).