Af594 conjugated goat anti mouse igg

HEK293T cells were seeded and cultured overnight on 3-aminopropyl triethoxysilane (APTES, Sigma) coated glass slides. The cells were fixed for 20 min in 3.7% formaldehyde (Fisher Scientific), permeabilized using 0.25% Triton X-100 and blocked with 4 × SSC/4%BSA for 1 h at room temperature. Rabbit antibodies against PGP9.5 (UCHL1) (Abcam) and mouse antibodies against TRF2 (Novus Biologicals) were incubated for 1 h and then for 1 h again at room temperature with secondary antibodies Alexa Fluor (AF) 488 conjugated goat anti-rabbit IgG and AF594 conjugated goat anti-mouse IgG, respectively (both Life technologies). Slides were then counterstained with DAPI (Sigma), mounted with Fluoromount G (Southern Biotech) and imaged with a Zeiss Z1 microscope using a 63x oil immersion objective with NA of 1.4 and image J software.

Cells were seeded and cultured overnight on 3-aminopropyl triethoxysilane (APTES, Sigma) coated glass slides. The cells were fixed for 20 min in 3.7% formaldehyde (Fisher Scientific), permeabilized using 0.25% Triton X-100 and blocked with 4x SSC/4%BSA for 1 h at RT. Primary antibodies for HMGA2 (D1A7) (rabbit monoclonal, Cell Signaling) and TRF2 (mouse monoclonal, Novus Biologicals) were incubated for 1 h at RT followed by 1 h incubation at RT with secondary antibodies such as Alexa Fluor (AF) 488 conjugated goat anti rabbit IgG and AF594 conjugated goat anti mouse IgG (both Life Technologies). Slides were then counterstained with DAPI (Sigma), mounted with Vectashield (Vector Laboratories, Burlington, ON) and imaged with a Zeiss Z1 microscope using a 63x oil immersion objective with NA of 1.4 and Axio Vision Software (Zeiss, Jena, Germany). Following deconvolution, colocalization analysis was performed using the ImageJ colocalization plugin on single, segmented nuclei as described previously [71 (link)]. The colocalizing signals were extracted and displayed as a separate image. 50 nuclei were randomly analysed under each experimental condition and the average number of colocalizing spots per nucleus was graphed with error bars representing standard errors of the means.