Eclipse e100080i microscope

Parasites growing in confluent HFFs were fixed in 4% paraformaldehyde for 10 min followed by washing in PBS three times. Fixed cells were incubated for 1 hr at room temperature in blocking buffer containing 3% 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 488/594/568/647 at room temperature for 1 hr. 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 CCD camera. Nikon NIS element software was used to analyze and capture images. Primary antibody dilutions were used as followed: rabbit α-HA (Cell Signaling) 1:1,000, rat α-HA (Roche), mouse α-MYC (Cell Signaling) 1:1,000, Toxoplasma α-Centrin-1 (Kerafast company) 1:2,000, rat α-IMC3 (supplied by Dr. Marc-Jan Gubbels) 1:2,000. For the visualization of brazyzoite tissue cyst walls, FITC-conjugated Dolichos biflorus lectin (Vector Laboratories) was used at a 1:500 dilution for 1 hr at room temperature as previously described (23) .