Immunofluorescence analysis of Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor® 488 conjugate was performed using A549 cells stained with alpha Tubulin (YL1/2) Rat Monoclonal Antibody (Product # MA1-80017). 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 primary antibody for 3 hours at room temperature. Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A-11006) 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: green). Nuclei (Panel b: blue) were stained with DAPI in SlowFade® Gold Antifade Mountant (Product # S36938). F-actin was stained with Rhodamine Phalloidin (Product # R415, 1:300) (Panel c: red). 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.