Alexa fluor 546 conjugated anti rat igg

Ileal PPs were dissected from the small intestine and transferred to a 10 cm dish containing 30 mL cold PBS. Excess intestinal tissues around the FAE were cut and removed under a stereomicroscope using forceps. PP’s were washed sufficiently by using a 1 mL syringe with a 26-gauge needle under a stereomicroscope. The mucous layer on the FAE should be flushed out with a water stream to prevent background noise detection. PPs were transferred to the 1.5 mL tube containing 1 mL PBS, washed by vortexing and the supernatant was discarded. After 3 washes, 300–1000 µL Cytofix/Cytoperm buffer (BD Biosciences, Franklin Lakes, NJ, USA) was used for blocking and permeabilization for 25 min at room temperature. Perm/wash buffer (BD Biosciences, Franklin Lakes, NJ, USA) was used to wash the PPs after which they were stained with PE-conjugated anti-GP2 antibody (MBL; 1:10 in Perm/wash buffer) overnight at 4 °C. Following the primary antibody staining, PPs were washed 3 times with wash/perm buffer and stained for 30 min at RT in Alexa Fluor 546-conjugated anti-Rat IgG (Thermo Fisher Scientific, Waltham, MA, USA). The PPs were washed again 3 times with wash/perm buffer and mounted with ProLong Diamond with Dapi mounting solution (Molecular Probes P36962). Slides were examined with a laser scanning confocal microscope (Zeiss LSM 800 LSCM).

Parasitized erythrocytes were smeared onto microscope slides and fixed in 4% formaldehyde/PBS for 10 mins, rinsed twice in PBS, treated with 0.03% triton/PBS for 10 mins, blocked with 1% BSA/PBS for 30 mins, then incubated with the following antibodies: 1:500 anti-Ty1 (Invitrogen), then 1:1000 Alexa Fluor 546-conjugated anti-rat IgG (Thermo Fisher Scientific); and/or 1:500 anti-HA (Roche), then 1:1000 Alexa Fluor 488-conjugated anti-rat IgG (Thermo Fisher Scientific). Slides were washed for 3 x 5 mins in PBS after each antibody step and in the penultimate wash 2µg/ml 4’,6-diamidino-2-phenylindole (DAPI) (Molecular Probes) was added. Slides were mounted with ProLong Diamond antifade mountant (Thermo Fisher Scientific) and imaged with a Zeiss LSM700 Confocal Microscope.