Goat anti rabbit igg conjugated with fitc

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.

Adult female I. scapularis 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 0.2M sodium cacodylate buffer, dehydrated in a graded series of ethanol and embedded in paraffin. 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% bovine serum albumin (BSA; Sigma-Aldrich) in PBS for 1 h at room temperature. The slides were then incubated for 14 h at 4°C with primary antibodies diluted 1:100 to 1:300 in 3% BSA/PBS and after 3 washes in PBS developed for 1 h with goat-anti-rabbit IgG conjugated with FITC (Sigma-Aldrich) (diluted 1:160 in 3% BSA/PBS). The slides were washed twice with TBS and mounted in ProLong Antifade reagent (Molecular Probes, Eugene, OR, USA) or in mounting medium containing DAPI (Vector Laboratories, Peterborough, UK). The sections were examined using a Leica SP2 laser scanning confocal microscope (Leica, Wetzlar, Germany). Sections of uninfected ticks and IgGs from preimmune serum were used as controls.

Immunofluorescence microscopy was used to investigate the presentation of recombinant proteins on surface display. A 250 µl of recombinant cells harboring Lpp-OmpA-PE and Non-OmpA-PE were harvested and centrifuged at 3500×g for 4 min and washed three times with PBS (pH 7.4) supplemented with 3% bovine serum albumin. In the next step, the cells were incubated with the rabbit anti-6His antibody diluted 1:1000 in PBS solution for 1 h at 4 °C. After washing five times with PBS, the cell-antibody complex was incubated 1.5 h at 4 °C with a goat anti-rabbit IgG conjugated with FITC (Sigma, USA) at a dilution of 1:500. For microscopic observation, cells were washed five times with PBS solution to remove unbound anti-6His-FITC antibody, then mounted on microscopic slides and was observed by fluorescence microscopy.