Immunofluorescence analysis of Chicken anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Product # A21201) was performed using SH-SY5Y (positive model) and T-47D (negative model) cells stained with Nestin Monoclonal Antibody (10C2), eBioscience™ (Product # 14-9843-80). 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. Chicken anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Product # A-21201, 1:2000 dilution) in 0.1% BSA in PBS for 45 minutes at room temperature, was used for detection of Nestin in the cytoskeleton (Panel a: Pink). Nuclei (Panel b: blue) were stained with Hoechst33342 (Product # H1399). F-actin was stained with Alexa Fluor® 488 Phalloidin (Product # A12379, 1:300) (Panel c: green). Panel d represents the composite image. The specificity of the secondary antibody was proved by the absence of signal in T-47D (negative model for Nestin) due to no primary antibody binding (Panel e). Non-specific staining was not observed with secondary antibody alone (panel f). The images were captured at 40X magnification in CellInsight CX7 LZR High-Content Screening (HCS) Platform (Product # CX7A1110LZR) and externally deconvoluted (D.Sage et al./Methods 115 (2017) 28–41).