Alexa fluor 488 conjugated goat anti mouse igg antibody

Fresh miracidia and cultured sporocysts were rinsed five times in CBSS+ on ice and fixed for 24 h in 1% PFA/1% Triton X-100 (Sigma–Aldrich) in sPBS at 4 °C. After fixation, larvae were washed five times in sPBS and once in blocking buffer (5% BSA/0.02% sodium azide), incubated in blocking buffer for 24 h at 4 °C and exposed to anti-rSmVAL9 (1:500 dilution in sPBS) for 24 h at 4 °C. Following primary antibody treatment, parasites were washed five times in sPBS and once in blocking buffer, and incubated in a secondary antibody mixture containing Alexa-fluor^®488-conjugated goat anti-mouse IgG (4 mg/ml), Hoechst 33258 dye (50 mg/ml) and Alexa-fluor^®546-conjugated phalloidin (7.5 mg/ml, Invitrogen) for 1 h at 22 °C in the dark. After five washes in sPBS, parasites were mounted on glass coverslips in Vectashield^® mounting medium (H-100, Vector Laboratories, Inc.) and imaged using an A1R laser scanning confocal microscope (Nikon Instruments Inc.) capable of scanning wavelengths of 408 nm, 488 nm and 561 nm for the excitation of Hoechst dye, Alexa Fluor^®488 and Alexa Fluor^®546, respectively. Negative controls included identically processed larvae in which: (i) NMS (1:500 diluted; Sigma–Aldrich) was substituted for the anti-SmVAL9 antibody and (ii) only the secondary Alexa-fluor^®488-conjugated goat anti-mouse IgG antibody was used.