Alexa fluor 488 goat anti mouse igg h l

Live, 7-day-old schistosomula were fixed in 4% paraformaldehyde (PFA) for 5 min at room temperature. The fixed parasites were incubated first with blocking buffer (1% bovine serum albumin (BSA) in PBS) for 1 h and then in serum (diluted 1:50 in blocking buffer) from either rSmPGM-immunized mice or control mice, for 1 h at room temperature. The worms were then washed with PBST before being incubated for 1 h in the dark at room temperature with Alexa Fluor^TM 488 goat anti-mouse IgG (H + L) (Invitrogen) diluted in blocking buffer at 1:100. The worms were again washed with PBST and then incubated with 0.3 mM 4’,6-diamidino-2-phenylindole (DAPI) in blocking buffer for 5 min, washed with PBST, mounted in Fluoromount, and placed in clear-bottom 96-well plates. The parasites were viewed using an inverted fluorescent microscope (Eclipse Ti, Nikon and Revolve, Echo).
Frozen sections (6 μm thick) of adult parasites in optimal cutting temperature (OCT) compound (Thermo Fisher Scientific) were hydrated with PBS and incubated with blocking buffer for 1 h at room temperature in a humidified chamber. The sections were processed for immunolocalization using the same techniques described above for whole, fixed schistosomula.

Serial sections (5 µm) of each block were adhered to poly-L-lysine-covered slides and incubated at 62 °C for 60 min. After deparaffinization and rehydration, the sections were heated in 10 mm citrate buffer (pH 6.0) for 15 min, and the slides were blocked with 10% goat serum in PBS for 1 h. The slides were washed with PBS and then incubated with the primary antibodies overnight. Alexa Fluor^TM 488 goat anti-mouse IgG (H + L) (Invitrogen; A11001) and Texas Red^®-X goat anti-rabbit IgG (H + L) (Molecular Probes; T-6391) were used as secondary antibodies. The slides were viewed under an LSM 980 confocal microscope (Carl Zeiss AG, Oberkochen, Germany).

Cells were seeded on the chamber slides and cultured with each reagent. Cultured cells were fixed with 4% paraformaldehyde phosphate buffer solution (Wako, Tokyo, Japan) for 15 min at room temperature and, subsequently, permeabilized in PBS containing 0.5% Triton-X. Chamber slides were blocked with 5% goat serum in PBS with 0.1% Tween 20 (BMS, Tokyo, Japan), followed by incubation with an anti-EREG antibody (AF1195, R&D Systems, Minneapolis, MN, USA) overnight at 4 °C. Alexa Fluor^TM 488 goat anti-mouse IgG (H + L) (Invitrogen, Waltham, MA, USA; A11001) was used for the secondary antibody. The images of cells were captured using BZ-X710 (Keyence, Osaka, Japan).