Transduced articular chondrocytes were cultured in eight-well chamber slides (Millipore) to test the expression of SV40LT and hTERT. Cells were washed with phosphate-buffered saline (PBS; Dako, Agilent Technologies Spain, Spain), fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 4% bovine serum albumin (all from Sigma-Aldrich). Subsequent incubation with two primary antibodies, mouse anti-SV40LT (SV40LT clone Pab 108; 1:100; Santa Cruz Biotechnology, USA) and rabbit anti-green fluorescent protein (GFP) labelled with Alexa Fluor 488 dye (A-21311; 1:500; Invitrogen), was performed at 4°C overnight.
After incubation with primary antibodies, cells were washed three times with PBS and incubated with a goat anti-mouse secondary antibody labelled with Alexa Fluor 594 dye (A-11032; 1:1,000; Thermo Fisher Scientific) at room temperature for one hour. After three additional washes in PBS, a two-minute incubation with Hoechst (bisBenzimide H 33342 trihydrochloride, Sigma-Aldrich) was performed. Slides were mounted with Glycergel aqueous mounting medium (Dako) and observed using an Olympus BX61 fluorescence microscope (Olympus Iberia, Spain) coupled to an Olympus DP70 digital camera (Olympus Iberia). Fluorescence micrographs were obtained employing the cellSens Dimension software (Olympus Iberia).
After incubation with primary antibodies, cells were washed three times with PBS and incubated with a goat anti-mouse secondary antibody labelled with Alexa Fluor 594 dye (A-11032; 1:1,000; Thermo Fisher Scientific) at room temperature for one hour. After three additional washes in PBS, a two-minute incubation with Hoechst (bisBenzimide H 33342 trihydrochloride, Sigma-Aldrich) was performed. Slides were mounted with Glycergel aqueous mounting medium (Dako) and observed using an Olympus BX61 fluorescence microscope (Olympus Iberia, Spain) coupled to an Olympus DP70 digital camera (Olympus Iberia). Fluorescence micrographs were obtained employing the cellSens Dimension software (Olympus Iberia).