Cy3 conjugated goat anti rabbit igg secondary antibody

HGF were grown on plastic coverslips (Thermo Fisher Scientific) of 13-mm diameter and placed in 24-well plates in presence and absence of glycine or trehalose for up to 60 min. Next, cells were washed twice with PBS and fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MI, USA) at pH 7.4 for 10 min. Subsequently, permeabilization of the cells was performed using 0.1% Triton X-100 (Sigma-Aldrich) for 5 min and then the cells were blocked with nonfat dry milk (Bio-Rad) for 1 h. After washing twice with PBS, cells were incubated with a rabbit anti-nuclear factor kappa B (NF)-κB p65 primary antibody (1:100, D14E12, Cell Signaling Technology, Danvers, MA, USA) at room temperature for 90 min. After the incubation step, cells were washed again twice with PBS and incubated with a CY3-conjugated goat anti-rabbit IgG secondary antibody (1:1000, ab6939, Abcam, Berlin, Germany) at room temperature for 45 min. The NF-κB p65 nuclear translocation was observed by using the ZOE™ Fluorescent Cell Imager (Bio-Rad). Untreated cells served as control.

HEK293 cells were seeded on 1% fibronectin‐coated 1.3 mm tissue culture coverslips (Sarstedt) in DMEM, 10% FBS. After plasmid transfection, cells were fixed for 10 min at room temperature with ice‐cold methanol, washed twice with PBS and incubated with 5% normal goat serum (NGS) before overnight incubation with polyclonal rabbit anti‐CSF‐1R (Invitrogen; PA5‐25974, 1:100) at 4°C. Cells were then washed twice with PBS and incubated with Cy3‐conjugated goat anti‐rabbit IgG secondary antibody (1:500; Abcam) for 2 h at room temperature and counterstained with Hoechst 33258 to visualise nuclei.
Macrophages were seeded in poly‐L‐lysine coated 8‐well chamber slides (Ibidi) at 2.8 × 10⁴ cells/cm². Macrophages were grown for 72 h, fixed in 4% PFA for 10 m at RT and stained as above with rat anti‐human CD68 (Abcam) primary antibody and 594‐conjugated goat ant‐rat IgG secondary antibody(Invitrogen).

Osteoblast-like cells were seeded on glass coverslips (Thermo Fisher Scientific Inc., Schwerte, Germany) and propagated until 70 % of confluence was achieved. Cells were treated with CAP as described above. After 15 min, 30 and 60 min cells were washed twice with 1x PBS (Invitrogen) and fixed with 4 % paraformaldehyde (Sigma-Aldrich) at pH 7.4 and room temperature for 10 min. After another washing step cells were permeabilized in 0.1 % Triton X-100 (Sigma-Aldrich) for 5 min. Next, the cells were washed again and blocked with 5 % BSA in PBS for 60 min to reduce the background staining. Afterwards, the cells were incubated with rabbit anti-p53 primary antibody (Abcam, Berlin, Germany; 1:200) at 4 °C overnight. Following another rinsing step, the cells were incubated with CY3-conjugated goat anti-rabbit IgG secondary antibody (Abcam; 1:1000) at room temperature for 45 min. Finally, location of p53 within the stained cells was analysed with the ZOE^™ Fluorescent Cell Imager (Bio-Rad). The images were captured with an integrated digital 5MP CMOS camera.

Cells from the adult RPE cell line ARPE-19 were obtained from the American Type Culture Collection (ATCC) (Manassas, VA). Dulbecco’s Modified Eagle’s Medium (DMEM): Nutrient Mixture F12, fetal bovine serum (FBS), bovine serum albumin (BSA), trypsin-EDTA, phosphate-buffered saline (PBS), Triton X-100, penicillin, streptomycin, and 4′, 6-diamidino-2-phenylindole (DAPI) were provided by Sigma-Aldrich (St. Louis, MO). Nunclon Δ-surface multidishes, pipettes, and other routine plastics were supplied by VWR International (West Chester, PA). Staurosporine and the primary rabbit anti-cleaved caspase-3 (Asp 175) antibody were obtained from Cell Signaling Technology (Danvers, MA). The Cy3-conjugated goat anti-rabbit IgG secondary antibody was purchased from Abcam (Cambridge, UK).