Immunofluorescence analysis of Growth-regulated alpha protein was performed using 70% confluent log phase A-375 cells treated with 10 ng/mL TNF alpha for 24 hours and untreated A-375 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 CXCL1 Polyclonal Antibody (Product # PA5-86508) at 1:100 dilution in 0.1% BSA, incubated at 4°C overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green) in A-375 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 showing cytoplasmic localization in TNF alpha treated A-375 cells. Panel e represents untreated A-375 cells that show no expression of CXCL1. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.