Immunofluorescence analysis of Calnexin was performed using 70% confluent log phase HeLa cells treated with Thapsigargin (1uM for 24hrs). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Calnexin Polyclonal Antibody (Product # PA1-30197) at 1:200 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 594 conjugate (Product # A-11062) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: red). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: green) was stained with Alexa Fluor™ 488 Phalloidin (Product # A12379, 1:300). Panel d represents the merged image showing Calnexin in the ER and cytoplasm. Panel e represents the untreated cells showing lower expression levels. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.