Immunofluorescence analysis of CALCOCO2 was performed using 70% confluent log phase HeLa cells treated with Thapsigargin 3uM for 48 hrs. 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 45 minutes at room temperature. The cells were labeled with CALCOCO2 Monoclonal Antibody (P1813) (Product # MA5-18293) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32723), (1:2000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Red) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Blue) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Cytosol localization. Panel e represents No signal Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.