Immunofluorescent analysis of V5 Tag was performed using 70% confluent V5-H3-6XHis transfected HEK-293 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with V5 Tag Monoclonal Antibody (E10/V4RR), DyLight 488 (Product # MA5-15253-D488) at 5 µg/mL and Histone H3 Recombinant Polyclonal Antibody (Product # 711055) at 0.5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Product # A32733) at a dilution of 1:2000 for 45 minutes at room temperature. Panel a (Green) represents V5 Tag. Panel b (Red) represents Histone H3. Panel c (Nuclei: Blue) represents ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). Panel d represents the merged image showing the co-localization of nuclear signals in transfected cells. Panel e represents untransfected HEK-293 cells. Panel f represents control cells with isotype control antibody to assess background. The images were captured at 60X magnification.

Immunofluorescent analysis of HeLa cells transfected with a construct containing a V5 Epitope Tag. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA (Product # 37525) for 15 minutes at room temperature. Cells were probed with a DyLight 488-conjugated V5 Epitope Tag monoclonal antibody (Product # MA5-15253-D488) at a dilution of 1:25 for at least 1 hour at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan or ToxInsight Instrument at 20X magnification.