Immunofluorescence analysis of TRAIL-R2 was performed using HCT 116 cells and HCT 116 treated with Thapsigargin (1 µM, 36 hours). The cells were fixed with 4% paraformaldehyde for 10 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with TRAIL-R2 Rabbit Monoclonal Antibody (Product # MA5-32693) at 1:100 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green) in HCT 116 treated cells. Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image of HCT116 treated cells, which shows higher expression for TRAIL-R2 protein showing localization in nucleus, cytoplasm and membrane. Panel e represents the merged image of untreated HCT 116 cells, that shows lower or no expression for TRAIL-R2 protein. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.