Mouse anti drosophila robo monoclonal antibodies

Uninfected and A. phagocytophilum-infected I. scapularis ISE6 tick cell slides were prepared using a cytocentrifuge. The slides were air-dried, fixed with ice-cold methanol for 10 min, then blocked with 3% BSA/PBS for 1 hr at RT. The slides were then incubated for 14 h at 4 °C with mouse anti-Drosophila Robo monoclonal antibodies (Creative Diagnostics, Shirley, NY, USA) diluted 1:64 in 3% BSA/PBS and, after 3 washes in PBS, developed for 1 h with goat anti-mouse IgG conjugated with FITC (Sigma-Aldrich) diluted 1:100 in 3% BSA/PBS. The slides were washed twice with PBS and mounted in ProLong Antifade with DAPI reagent (Molecular Probes, Eugene, OR, USA). The sections were examined using a Zeiss LSM 800 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) with a 63x oil immersion objective. Using ImageJ, an outline was drawn around each cell and area, mean fluorescence and integrated density were measured, along with several adjacent background readings. Then, the total cytoplasmic corrected cellular fluorescence (TCCF) was calculated as integrated density − (area of selected cell × mean fluorescence of background readings)^{59 (link)} and compared between infected and uninfected cells by Student’s t-test with unequal variance (p = 0.05; N = 10 biological replicates).

Twenty-five μg of protein lysate from uninfected and A. phagocytophilum-infected I. scapularis ISE6 tick cells were methanol/chloroform precipitated, resuspended in Laemmli sample buffer and separated on a 12% sodium dodecyl sulfate (SDS) polyacrylamide precast gel (ClearPage Expedeon, VWR, Radnor, PA, USA). After electrophoresis, proteins were transferred to a nitrocellulose blotting membrane (GE Healthcare Dharmacon Inc.), blocked with 3% BSA (Sigma) in Tris-buffered saline (TBS; 50 mM Tris-Cl, pH 7.5, 150 mM NaCl) and incubated overnight at 4 °C with mouse anti-Drosophila Robo monoclonal antibodies (Creative Diagnostics) or rabbit anti-Cytochrome c (Cyt c) antibodies (H-104: sc-7159; Santa Cruz Biotechnology, Inc. Dallas, TX, USA)^{3 (link)} diluted 1:100 in 3% BSA/PBS. Cyt c was included as a control because protein levels are not affected by A. phagocytophilum infection of ticks and ISE6 cells^{3 (link),19 (link)}. For Cyt c, proteins from human HL60 cells were included as a positive control^{3 (link)}. To detect the IgG antibodies bound to tick proteins, membranes were incubated with goat anti-mouse or anti-rabbit IgG peroxidase antibody (Sigma) diluted 1:1000 in 3% BSA/PBS. Immunoreactive proteins were visualized with chemiluminescence with Pierce ECL Western Blotting Substrate (Thermo Scientific).