Immunofluorescence analysis of VPS35 was performed using U-87 MG cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with VPS35 Goat Polyclonal Antibody (Product # PA5-18248) at 5µg/mL in 0.1% BSA and incubated overnight at 4 degree and then labeled with Rabbit anti-Goat IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A-11078) 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 composite image showing endosomal localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.