Fitc conjugated dolichos biflorus lectin

The indirect immunofluorescence assay (IFA) was performed as described previously [25 (link)]. Parasites were inoculated onto glass coverslips with confluent monolayers of HFF cells and incubated at 37°C. After washing with PBS three times, the coverslips were fixed and permeabilized with 4% formaldehyde-0.2% Triton X-100 in PBS, pH 7.0, for 15 min. After washing with PBS three times, samples were blocked with blocking solution (3% bovine serum albumin (BSA) fraction V in PBS, pH 7.0) for 20 min. Samples were then incubated for 1 hour with primary antibodies diluted 1:500 in blocking solution. Anti- LDH1/2, IMC1 and SAG1 antibodies [16 (link)] were used. After washing three times with PBS, samples were incubated for 1 hour with goat anti-mouse or anti-rabbit secondary antibody conjugated with Alexa fluor 488 or 594 diluted 1:1000 in blocking solution. For cyst wall staining, FITC-conjugated Dolichos biflorus lectin (Vector laboratories Inc., CA, USA) was used. After washing three times with PBS and once with water, samples were mounted with Mowiol (9.6% Mowiol, 24% glycerol, 100 mM Tris [pH 8.5]) and examined using a Leica TCS NT Confocal Laser Scanning Microscope (Leica Microsystems, Wetzlar, Germany).

Parasites growing in confluent HFF cells were fixed in 4% paraformaldehyde for 10 min followed by washing in PBS three times. Fixed cells were incubated for 1 h at room temperature in blocking buffer containing 3% bovine serum albumin (BSA) and 0.2% Triton X-100. The cells were incubated with the designated antibodies in blocking buffer at 4°C overnight followed by washing in PBS. The cells were then incubated with secondary antibodies coupled to Alexa Fluor 488/594/568/647 at room temperature for 1 h. The cells were finally washed with PBS and mounted with ProLong gold antifade mounting solution (Invitrogen) containing DAPI (Invitrogen) and then visualized using a Nikon Eclipse E100080i microscope. Images were captured with a Hamamatsu C4742‐95 charge-coupled-device (CCD) camera. Nikon NIS element software was used to analyze and capture images. Primary antibody dilutions were used as follows: rabbit anti-HA (Cell Signaling) 1:1,000, rat anti-HA (Roche) 1:1,000, mouse anti-MYC (Cell Signaling) 1:1,000, Toxoplasma anti-Centrin-1 (Kerafast Inc.) 1:2,000, rat anti-IMC3 (supplied by Marc-Jan Gubbels) 1:2,000. For the visualization of bradyzoite tissue cyst walls, fluorescein isothiocyanate (FITC)-conjugated Dolichos biflorus lectin (Vector Laboratories) was used at a 1:500 dilution for 1 h at room temperature as previously described (23 (link)).