Anti rabbit igg fitc

Purified qCD4s were incubated with gp120 (250 ng/ml) or AT-2-inactivated HIV-1. Immunofluorescence was performed by serial staining with goat anti-CD4 polyclonal Abs (R&D Systems), Cy3-conjugated secondary Abs (Sigma), rabbit anti-gp120 antiserum (ABI), anti-rabbit IgG-FITC (Dako), biotinylated anti-CXCR4 monoclonal Abs (R&D Systems), and streptavidin-Qdot 605 (Life Technologies, Carlsbad, CA), in that order. The cells were then fixed with 4% paraformaldehyde. sICs were visualized by staining purified rCD4s with goat anti-CD4 polyclonal Abs, Cy3-conjugated secondary Abs, biotinylated F(ab′)² anti-human Igs (Life Technologies), and streptavidin-Qdot 525, in that order. Multicolor confocal and DIC images with a 512×512 resolution were acquired using a Zeiss LSM510 system with a Plan-Apochromatic 63×1.4 NA oil immersion DIC objective (Carl Zeiss, Oberkochen, Germany) using multi-track scanning.

10⁶lpg1-/- and WT parasites were washed with PBS and incubated in M199 medium containing 10 µg/ml CRP, control media only or media with CRP and 15 mM EDTA for 1 hour at room temperature. Parasites were centrifuged and fixed in 4% (w/v) PFA, washed and 2.5 x 10⁵ parasites, air dried onto slides. Slides were rehydrated and stained with rabbit anti human CRP (Calbiochem) followed by anti-rabbit IgG FITC (Dako) both for 1 hour with washing between stages. The slides were dried and counterstained with vectorshield + DAPI and coverslips attached. Slides were visualised using the Zeiss LSM880 confocal microscope.