Immunofluorescence analysis of HSP40 was performed using 70% confluent log phase HeLa cells heat shocked at 43°C for 1 hr. 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 HSP40 Polyclonal Antibody (Product # PA5-17382) at 1:400 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® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). 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 showing nuclear translocation upon heat shock. Panel e shows untreated HeLa cells with cytoplasmic localization. Panel f represents control cells with no primary to assess background. The images were captured at 60X magnification.