Alexa 488 conjugated goat anti human igg h l

For immunofluorescence experiments, PBMC from 5 NP, 5 LTNP and 2 HD were incubated with the following antibodies: mouse monoclonal anti-LC3B (1:50, Cosmo Bio, CTB-LC3-1-50); rabbit polyclonal anti-TRIM5α (1:50, Novusbio NBP1-76601), human monoclonal anti-gp120 (kindly provided by Dr. Jean-Luc Perfettini, Paris, France); mouse monoclonal anti-HIV-1 Nef (1:50, Abcam ab42355).
Sections were thoroughly rinsed with PBS, then incubated for 1 h at RT with 1:400 Alexa 594 conjugated goat anti-rabbit IgG (Invitrogen, Rockford, IL, USA, A-11037); 1:400 Alexa 488 conjugated goat anti-mouse IgG (H + L) (Invitrogen, Life Technologies, A-11029), Alexa 647 1:400 goat anti-rabbit IgG (Invitrogen, Life Technologies, A32733), 1:400 Alexa 488 conjugated goat anti-human IgG (H + L) (Invitrogen, Life Technologies, A-11013). Controls were performed by omitting the primary antibodies. Slides were observed and photographed in a Leica TCS SP2 confocal microscope (Leica Microsystems GmgH, Ernst-Leitz-trasse 17-37 35578 Wetzlar, Germany).
The percentage of colocalizations between gp120-positive dots, with LC3-positive dots and TRIM5α-positive dots with respect to the total gp120-positive dots was evaluated.
A minimum of 30 cells/patient were observed. Cell counting was performed by 3 independent researchers; data are presented as mean ± S.D.

To assess the binding of Fc-Proteins to virus particles, HFFs were seeded on gelatin-coated 8-well μ-slides (Ibidi) at a density of 4 x 10⁴ cells per well one day prior to infection. Virus preparations were preincubated with Fc-fusion proteins at indicated concentrations for 2 h at 37°C and subsequently precooled on ice for 15 min. Medium of precooled cells was exchanged against virus mixtures and incubated on ice for 90 min. The cells were washed once with MEM5 prior to fixation with 80% acetone. For staining of viral particles, cells were incubated with a mouse monoclonal antibody recognizing the abundant viral protein pUL32 (generously provided by W. Britt) [73 (link)]. As a secondary antibody, Cy3-conjugated goat anti-mouse IgG F(ab’)₂ (Jackson ImmunoResearch) was used. Visualization of bound Fc-proteins was achieved by applying Alexa488-conjugated goat-anti-human IgG (H+L; Invitrogen). For better orientation, cell nuclei were stained with DAPI. For quantification of PDGFR-alpha-Fc binding to HCMV particles, the grey values of 100 particles per condition were analyzed using AxioVision Software (Zeiss).