A ring-stage adhesion assay was conducted as described above, and after the final gravity wash, slide chambers were removed per the manufacturer's instructions. The slides were fixed in ice-cold acetone-methanol (90:10) and allowed to return to room temperature for 30 min. Primary incubation was with 50 μl/well of IT4var09 anti-NTS-DBL1α and anti-DBL2γ and negative controls (nonimmunized rabbit IgG and antibody to an irrelevant PfEMP1 variant, NTS-DBL1α of TM180var1 [28 (link)]). One mg/ml total IgG stock of each antibody was diluted 1:5,000 in PBS–1% BSA to give a final concentration of 0.2 μg/ml, and incubation was for 1 h at room temperature in a humidified box. Cells were washed 3 times with 50 μl PBS/well for 5 min each time, and then the secondary antibody (Alexa Fluor 488 goat-anti-Rabbit IgG; A-11034; Invitrogen) was added at 1:1,000 dilution in PBS–1% BSA containing 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI) to stain parasite nuclei and incubated for 45 min as described above. A final wash was conducted (50 μl per well for 5 min each wash) before mounting with 1.25 mg/ml DABCO (1,4-diazabicyclo[2.2.2]octane) in 50% glycerol–50% PBS (29 (link)).
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