Fitc conjugated dolichos biflorus agglutinin dba fitc

Indirect immunofluorescence assays were performed according to previously described protocols (42 (link)). The following primary antibodies were used: mouse anti-HA (Medical & Biological Laboratories Co., Japan), rabbit anti-HSP60, and rabbit anti-TgALD antibodies. Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies (Life Technologies, USA), Hoechst 33342 (Beyotime, China), and FITC-conjugated Dolichos biflorus agglutinin (DBA-FITC) (Vector Laboratories, USA) were deployed as indicated to visualize the signals. Parasites were imaged with the FV1000 LSCM confocal laser scanning microscope (Olympus, Japan).

The plaque and replication assays to determine the overall growth and proliferation fitness of tachyzoites were performed following the protocols reported previously (41 (link)). To evaluate the bradyzoite differentiation efficiencies of the ME49 and ME49Δmpc1 strains, parasites were cultured for 3 days in alkaline RPMI 1640 medium supplemented with 1% FBS and 50 mM HEPES (pH 8.2, ambient CO₂). Samples were fixed by 4% paraformaldehyde, permeabilized in 0.1% Triton X-100 (Sigma-Aldrich, USA), and then stained with Hoechst 33342 (Beyotime, China), FITC-conjugated Dolichos biflorus agglutinin (DBA-FITC; Vector Laboratories, USA), and rabbit anti-ALD (fructose-bisphosphate aldolase) antibody (provided by David Sibley, Washington University, St. Louis, MO, USA). The formation of bradyzoites was calculated by the ratio of DBA-FITC-positive versus ALD-expressing vacuoles.

To stain tachyzoites, extracellular parasites were used to infect fresh HFF monolayers seeded on glass coverslips and cultured for 24 h. Then the samples were stained with periodic acid–Schiff (PAS) [26 (link)]. To stain bradyzoites, parasite cultures were subjected to alkaline stress (culture medium with pH = 8.2, ambient CO₂) for 5 days in T25 flasks. Subsequently, syringe-released parasites were used to infect fresh HFF cells on coverslips and cultured under the same alkaline conditions for 4 days prior to staining [19 (link)]. All samples were fixed with 4% paraformaldehyde, permeabilized with Triton X-100 (Sigma-Aldrich, USA) and stained with Hoechst 33342 (Beyotime, China) and/or FITC-conjugated Dolichos biflorus agglutinin (DBA-FITC) (Vector Laboratories, USA). Successive PAS staining was performed by a standard procedure reported previously [19 (link)]. Briefly, samples were incubated in 1% periodic acid (Sigma-Aldrich, USA) for 5 min, washed with tap water for 1 min, and rinsed once with distilled water. Cells were then incubated with Schiff's reagent (Sigma-Aldrich, USA) for 15 min, washed with tap water for 10 min, and rinsed three times with PBS. Cultures were imaged by the Olympus BX53 microscope (Olympus, Japan) equipped with an Axiocam 503 mono camera (Carl Zeiss, Germany).