Alexa fluor 488 anti rabbit igg h l

Worms were embedded in OCT compound and then frozen in liquid nitrogen. A cryostat was used to generate adult parasite frozen sections (7 μm thick) and these were fixed in ice-cold acetone for 30 min at −20 °C. CHO cells transfected with plasmid encoding SmaRT were fixed in 4% paraformaldehyde for 20 min, 48h after transfection. For immunolocalization, parasite sections and CHO cells were first washed ×3 in PBS. They were then incubated in PBS containing 1% BSA (blocking buffer) for 1h. Primary, purified anti-SmaRT, antibody at 1:100 dilution was next applied to the samples for 1h. Following this, samples were washed ×3 in PBST before being incubated for 1h with Alexa Fluor-488-anti-rabbit IgG (H + L, Invitrogen) at 1:100 dilution in blocking buffer, as in previous immunolocalization work [24 ]. Samples were again washed in PBS before being mounted in Fluoromount and examined by inverted fluorescent microscopy using a TH4–100, Olympus microscope. To stain nuclei, sections were incubated with DAPI (0.3 μM) for 5 min. To stain actin, some sections were incubated with phalloidin (1 unit mixed with the secondary antibody, Invitrogen) for 1h.