Goat anti rabbit igg conjugated with phycoerythrin pe

Adult I. scapularis females were infected with A. phagocytophilum (NY18) as described above. Female ticks were removed from the sheep 10 days after infestation, held in the humidity chamber for 4 days and fixed with 4% paraformaldehyde in 0.2 M sodium cacodylate buffer, dehydrated in a graded series of ethanol and embedded in paraffin (Ayllón et al., 2015 (link)). Sections (4 μm) were prepared and mounted on glass slides. The paraffin was removed from the sections with xylene and the sections were hydrated by successive 2 min washes with a graded series of 100, 95, 80, 75, and 50% ethanol. The slides were treated with Proteinase K (Dako, Barcelona, Spain) for 7 min, washed with PBS and incubated with 3% BSA (Sigma-Aldrich) in PBS for 1 h at RT. The slides were then incubated for 14 h at 4°C with primary rabbit IgG antibodies diluted 1:100 in 3% BSA/PBS and, after 3 washes in PBS, developed for 1 h with goat-anti-rabbit IgG conjugated with phycoerythrin (PE) (Sigma-Aldrich) (diluted 1:50 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). Sections of uninfected ticks and IgG from pre-immune and anti-ISE6 sera were used as controls.

Adult I. scapularis females were fed on uninfected and A. phagocytophilum (NY18)-infected sheep as described before^{66 (link)}. Infected and uninfected female fed ticks were fixed, embedded in paraffin, and sections (4 μm) prepared and mounted on glass slides as previously described^{36 (link)}. The slides were processed^{36 (link)} and then incubated for 14 h at 4 °C with anti-ubiquinol-cytochrome c reductase core protein I antibodies (ab96333; Abcam, Cambridge, UK) diluted 1:100 in 3% BSA/PBS and after 3 washes in PBS, developed for 1 h with either goat-anti-rabbit IgG conjugated with phycoerythrin (PE) (Sigma-Aldrich) 1:50 dilution in 3% BSA/PBS or goat anti-rabbit IgG conjugated with FITC (Sigma-Aldrich) 1:200 dilution in 3% BSA/PBS. Finally, the slides were mounted in ProLong Antifade with DAPI reagent (Molecular Probes) and examined using a Zeiss LSM 800 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) with x40 oil immersion objectives.