Immunofluorescence studies with P. falciparum trophozoites that were treated with 10 μg/mL OZ277 for 2 h. (a) In immunofluorescence colocalization studies, the blood smears were fixed with 5% formaldehyde and 0.01% glutaraldehyde, permeabilized with 0.5% Triton-X-100, and blocked with 1% BSA in PBS for 1 h. The primary antibodies used were directly labeled OZH04-2/2 (Alexa 488, green signal) and GAPDH (Alexa 594, red signal). For both, the exposure time was 2 s. Nuclei were stained with DAPI (blue signal, exposure time of 0.05 s). The bottom row left shows the merge of OZH04-2/2, GAPDH, and DAPI and the bottom row right, the reference image (exposure of 1 s). Scale bar = 2 μm. (b) Same as (a), except that unlabeled primary antibodies OZH04-2/2 were used. After six washes with PBS, 20 μg/mL of the secondary antibody goat antimouse Alexa 488 (green signal) was incubated for 1 h at room temperature. The exposure time was 0.1 s. Nuclei were stained with DAPI (blue signal, exposure time of 0.05 s). The bottom row left shows the merge of OZH04-2/2 and DAPI, and the bottom row right shows the merge of OZH04-2/2 and the reference image (exposure of 1 s). Scale bar = 2 μm. (c) Same as (b), except that the secondary antibody used was goat antimouse Alexa 594 with an exposure of 1 s (red signal).
Jourdan J, Matile H, Reift E, Biehlmaier O, Dong Y, Wang X, Mäser P, Vennerstrom JL, Wittlin S. Monoclonal Antibodies That Recognize the Alkylation Signature of Antimalarial Ozonides OZ277 (Arterolane) and OZ439 (Artefenomel). ACS Infect Dis. 2016 Jan 8;2(1):54-61.