Rat monoclonal α ha clone 3f10

Confluent HFF cultures on glass coverslips were infected with parasites for the indicated times and under specified growth conditions. Monolayers were fixed, permeabilized, and incubated with antibody as previously described (51 (link)). The following primary antibodies were used: rat monoclonal α-HA (clone 3F10; Roche Applied Sciences), rabbit α-Myc (clone 71D10; Cell Signaling Technology), mouse α-Centrin (clone 20H5; Millipore Sigma), mouse α-CenH3 (centromere marker; kindly provided by Boris Striepen, University of Pennsylvania, Philadelphia, PA), mouse α-Ndc80 (outer kinetochore marker; kindly provided by Marc-Jan Gubbels, Boston College, MA), rabbit α-MORN1 (centrocone and basal complex stains; kindly provided by Marc-Jan Gubbels, Boston College, MA), and rabbit and mouse α-IMC1 (parasite shape and internal daughter bud strains; kindly provided by Gary Ward, University of Vermont, VT). All Alexa-conjugated secondary antibodies (Molecular Probes, Life Technologies) were used at a dilution of 1:500. Coverslips were mounted with Aquamount (Thermo Scientific), dried overnight at 4°C, and viewed on a Zeiss Axiovert microscope equipped with a 100× objective using the ApoTome slicer. The collected images were processed first using Zeiss Zen software and then in Adobe Photoshop 2020 using linear adjustment when needed.

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.