Rhodamine labeled dolichos biflorus agglutinin

Tachyzoites were differentiated in vitro into bradyzoites within cysts essentially as previously and elegantly described by Tobin and colleagues (58 (link)). Differentiation media contained Roswell Park Memorial Institute medium (RPMI) without bicarbonate supplemented with 2.05 mM l-glutamine (Hyclone), 20 mM HEPES-free acid (IBI Scientific), 1% XL-glutamine (a long-lasting stable form of glutamine; VWR), 1% FBS, and 1% penicillin-streptomycin. The pH of differentiation medium was adjusted to 8.1 with sodium hydroxide and filter sterilized. HFF cells were cultured on circular microcover glass, and confluent monolayers were infected with type II parasites at an MOI of ~0.5. Three hours after infection, the infected cells were washed once in DPBS supplemented with Ca²⁺ and Mg²⁺, and cultures were incubated in differentiation media for 3 days at 37°C in ambient air. Infected cells were fixed in 4% paraformaldehyde, and the excess was quenched with 0.1 M glycine. All samples were permeabilized and blocked in 3% FBS plus 0.2% Triton X-100 for 30 min at room temperature, and then they were incubated with a 1:250 dilution of rhodamine-labeled Dolichos biflorus agglutinin (Vector Laboratories) for 1 h at RT. The preparations were washed three times with DPBS, mounted in SlowFade gold antifade with DAPI (Life Technologies), and imaged by confocal microscopy.

Immunofluorescence (IF) and RNA in situ hybridization were performed on 5 μm cryosections using standard procedures. DIG-labeled (Roche) riboprobes were generated from pCRII-TOPO plasmid (ThermoFisher Sci) with mLRH-1 cDNA corresponding to bases 595–1683. Antibodies against hLRH-1 (1:200, Sigma HPA005455), FLAG (1:300, Sigma F7425), cleaved Caspase-3 (1:1000 (WB) and 1:400 (IF), Cell Signaling 5A1E), CD-44 (1:500, Tonbo 70-0441), lysozyme (1:200, DAKO EC 3.2.1.17) and MUC-2 (1:300, Santa Cruz Biotechnology sc-15334) were used with Alexa Fluor-conjugated secondary antibodies 1:300 (Millipore, Invitrogen). For Goblet staining, Rhodamine-labeled Dolichos Biflorus Agglutinin (Vector Labs) was used at 1:200 dilution.