Monolayers of HFF cells were grown on coverslips and infected with parasites under indicated conditions. Cells were fixed in 4% PFA, permeabilized with 0.25% Triton X-100, blocked in 1%BSA and incubated sequentially with primary and secondary antibody [16 ]. The following primary antibodies were used: mouse monoclonal α-ISP1 (clone 7E8) [46 ] and α-Atrx1 (clone 11G8) (kindly provided by Dr. Peter Bradley, UCLA) [75 (link)], α-TgCenH3 [23 ] (kindly provided by Dr. Boris Striepen, University of Georgia, Athens), α-acetylated alpha Tubulin (Abcam), rat monoclonal α-HA (clone 3F10, Roche Applied Sciences), rabbit polyclonal α-myc (Cell Signaling Technology), α-Human Centrin 2 [17 (link)], α-MORN1 (kindly provided by Dr. Marc-Jan Gubbels, Boston College) [27 (link)] and α-IMC1 (kindly provided by Dr. Gary Ward, University of Vermont). Alexa-conjugated secondary antibodies of the different emission wavelengths (Molecular Probes, Thermo Fisher Scientific) were used at a dilution of 1:1000. Stained parasites on the coverslips were mounted with Aqua-mount (Lerner Laboratories), dried overnight at 4°C, and viewed on a Zeiss Axiovert Microscope equipped with 100x objective. Images were collected and processed using Zeiss Zen software and were further processed in Adobe Photoshop CC using linear adjustment when needed.
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