Alexa fluor 488 conjugated goat anti human igg

50 μl of sera diluted in PBS with 2% BSA were added to each well of the 96-well plate containing air-dried PfSPZ. Sera samples were added at twofold dilutions starting at 1:50. After adding samples, plates were incubated at 37 °C for 1 h. Plates were washed in PBS three times on an Aquamax Microplate washer. Alexa Fluor 488 conjugated goat anti-human IgG (Molecular Probes) was diluted to 1:250 in PBS with 2% BSA and 50 μl was added to each well. The plates were then incubated for 1 h at 37 °C. Plates were washed three times with PBS on an Aquamax Microplate washer. 100 μl PBS was added to each well. The plates were sealed using a plate sealer and stored in the dark at 4 °C until data acquisition. Samples were assessed by scanning the plates using an Acumen eX3 laser scanning imaging cytometer. The positive control was pooled human serum taken two weeks after the last immunization from 12 uninfected volunteers immunized 4 or 5 times with 1.35 × 10⁵ PfSPZ Vaccine^{5 (link)}. The Acumen image cytometer scans the entire surface area of each well in a 96-well plate and the fluorescence intensity values (arbitrary units) therefore represent the signal from all 5 × 10³ PfSPZ that were seeded in each well.

For Figure 1, ANA staining patterns of TIN mAbs were visualized using an IIF kit (INOVA diagnostics). For subsequent studies, HEp-2 cells were fixed in methanol [17 (link)] and incubated with mAbs (50 μg/ml), followed by Alexa Fluor 488-conjugated goat anti-human IgG (2 μg/ml) and Hoechst 33342 (both Life Technologies). Selected slides were sequentially stained with anti-vimentin V9 (2 μg/ml, DAKO) or anti-giantin (1μg/ml, Abcam) followed by Alexa Fluor 568-conjugated donkey anti-mouse antibodies (2 μg/ml, Molecular Probes). Samples were imaged using a Leica SP5 II STED-CW laser scanning confocal microscope.