Immunofluorescence analysis of F(ab')2-Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Product # A-11020) was performed using HeLa cells stained with alpha Tubulin (Product # 236-10501) Mouse Monoclonal Antibody (Product # A11126). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, blocked with 1% BSA for 1 hour and labeled with 2 µg/mL of mouse primary antibody for 3 hours at room temperature. F(ab')2-Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Product # A-11020) was used at a concentration of 4 µg/mL in phosphate buffered saline containing 0.2 % BSA for 45 minutes at room temperature, for detection of alpha Tubulin in the cytoplasm (Panel a: red). Nuclei (Panel b: blue) were stained with DAPI in SlowFade® Gold Antifade Mountant (Product # S36938). F-actin was stained with Alexa Fluor® 488 Phalloidin (Product # A12379, 1:300) (Panel c: green). Panel d represents the composite image. No nonspecific staining was observed with the secondary antibody alone (panel f), or with an isotype control (panel e). The images were captured at 60X magnification.