Carl epifluorescence microscope

Trypan blue dye (Cat. # 1450021; BioRad, Hercules, CA, USA) exclusion tests were carried out using a TC20 automated cell counter (Bio-Rad, Hercules, CA, USA). Phase contrast images of the cells were visualized using a 20× objective lens on Carl Zeiss epifluorescence microscope (Zeiss, Thornwood, NY, USA) and captured via a charge-coupled device (CCD) camera fitted to the microscope. The Zen Blue Lite software (Carl Zeiss Microscopy GmbH, Jena, Germany) was used to process the images.

Cultured T. solium cysticerci were washed with PBS 1× and embedded in Tissue Tek (Triangle Biomedical Science, Arizona, USA), and immediately frozen at -80 °C until use. Parasitic tissue sections (5 μm) were fixed with frozen acetone for 10 min, washed 3 times in PBS-Tween 0.3% and blocked for 1 h with PBS containing 1% bovine albumin. Cross-sections were then incubated with a 1:100 dilution of PGRMC polyclonal antibody obtained from T. spiralis PGRMC (cloned, sequenced, synthesized and produced by Dr. Romel Hernández-Bello, who kindly donated it to us) for 45 min at 37 °C, washed with PBS and then incubated with Alexa 488-conjugated rabbit anti-mouse antibody (Invitrogen, California, USA) at 1:300 dilution. Control experiments were assessed incubating the 5 μm thick tissue sections in the presence of Alexa 488-conjugated rabbit anti-mouse antibody alone at the same dilution. To eliminate background fluorescence, samples were contrasted with 0.025% Evans Blue for 10 min. After two single washings, samples were mounted in Vectashield mounting medium (Vector Laboratories Inc.,Boston, USA) and examined with a Carl Zeiss epifluorescence microscope (Carl Zeiss, Berlin, Germany).