Alexa 488 conjugated goat anti mouse 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 confirm the localization of tyrosinated tubulin and detyrosinated tubulin, slides of 25 μm-sections were blocked with 5% donkey serum for an hour, followed by the dual-labeled immunofluorescent staining. The following primary antibodies were used: mouse monoclonal anti-tyrosinated tubulin (1:1000) and rabbit polyclonal anti-detyrosinated tubulin (1:400). After rinsing in PBST (phosphate-buffered saline, 1% Tween-20), the slides were incubated with the appropriate secondary antibodies diluted 1:200 in PBS as follows: Alexa 488-conjugated goat anti-mouse IgG (H + L) (Invitrogen, A-11029), and Alexa 647-conjugated donkey anti-rabbit IgG (H + L) (Jackson ImmunoResearch, 711–605-152). Rhodamine-conjugated PNA at 1:2000 dilution in PBS was used to detect acrosomes (Vector Laboratories, RL-1072) as described previously [19 (link), 27 (link), 28 (link)], counterstained with DAPI, and mounted with Glycergel mounting medium. Slides were first viewed on a Nikon photomicroscope under fluorescence optics. Fluorescent images were captured using a confocal Zeiss LSM710 microscope as described previously [33 (link), 34 (link)]. Z stacks, composed of 0.25-μm optical sections, were obtained using the Zen 2011 software (Zeiss). Images were subsequently analyzed and processed using the public domain software ImageJ (NIH).

Mouse monoclonal anti-nucleolin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, C23 (MS-3), sc-8031), mouse monoclonal anti-β-actin antibody (Sigma, AC-15), mouse monoclonal anti-Flag antibody (ANTI-FLAG M2 Monoclonal Antibody, Sigma, F1804), mouse monoclonal anti-HP1γ antibody (Sigma, MAB3450), mouse monoclonal anti-RNA polymerase I antibody (Santa Cruz Biotechnology, anti-RPA194 antibody (C-1), sc-48385), rabbit polyclonal anti-GFP antibody (Santa Cruz, sc-8334), goat anti-rabbit IgG-HRP (Santa Cruz, sc-2054), goat anti-mouse IgG-HRP (Santa Cruz #sc-2005), Alexa 568-conjugated goat anti-mouse IgG (H+L) (Thermo Fisher Scientific, Waltham, MA, USA, A11004), Alexa 488-conjugated goat anti-mouse IgG (H+L) (Thermo Fisher, A11029), and Alexa 568-conjugated goat anti-rabbit IgG (H+L) (Thermo Fisher, A11036), mouse monoclonal anti-dimethylated histone H3 lys36 antibody (Active Motif, California, USA, MABI0332), and rabbit polyclonal anti-histone H3 antibody (Abcam, Cambridge, UK, ab1791), were purchased. The anti-KDM2A antibody was described previously [19 (link)]. The anti-Fbxl11 (KDM2A) antibody (Abcam, ab99242) was purchased and used to detect the C-terminal half of KDM2A.