Goat anti rabbit igg conjugated with alexa 488 green

Freshly lysed parasites were syringed, filter purified, and resuspended at 5 × 10⁷ parasites per mL in invasion medium (DMEM supplemented with 3% cosmic calf serum). Two hundred microliters of the parasite resuspension were inoculated into each well of an 8-well chamber slide preseeded with HFF cells, and parasites were allowed to invade host cells for 30 min before fixation with 4% formaldehyde for 20 min. Slides were immunostained with mouse anti-TgSAG1 monoclonal antibody (1:2000) for 1 h to label attached parasites followed by a secondary stain using goat anti-mouse IgG conjugated with Alexa 594 (red) (Invitrogen, 1:1000). Next, the slide was permeabilized with 0.1% Triton X-100 for 10 min, and then stained with a rabbit polyclonal anti-TgMIC5 antibody (1:1000) and goat anti-rabbit IgG conjugated with Alexa 488 (green) (Invitrogen, 1:1000) to label all parasites, including invaded and attached parasites. DAPI was also included for nuclear staining. Six fields of view for each strain were captured by a Leica DMi8 inverted epifluorescence microscope, and ImageJ software was used for analysis. The following equation was used to calculate the invasion efficiency of each strain: [(sum of green parasites) − (sum of red parasites)] ⁄ total host nuclei. The assay was repeated, at minimum, in three biological replicates.