A-MLV Env co-localization with cholesterol, GM1 and cav-1. A) A-MLV Env co-localization with cholesterol. NIH3T3 cells producing wild-type A-MLV were treated with filipin for cholesterol detection (left column) and with an A-MLV Env specific antibody (second column) after fixation and treatment with PBS (top) or TX-100 at 4°C (bottom). Co-localization result in pink spots (merged images, third column). The column on the right shows the result of the co-localization finder plugin of the ImageJ program  merged with the original A-MLV Env staining. Turquoise colour indicates co-localization of A-MLV Env with cholesterol. B) A-MLV Env and cav-1 co-localization monitored by fluorescence microscopy. Immunofluorescent detection of cav-1 (left) and the A-MLV Env (middle) after treatment with TX-100 at 4°C in NIH3T3 cells producing A-MLV. Co-localization result in yellow spots (right). C) A-MLV Env (left) and GM1 (middle) were detected by immunofluorescence in A-MLV producing NIH3T3 cells after PBS (top) or TX-100 treatment at 4°C (bottom). Co-localization result in yellow spots (right). All photographs were taken using a fluorescence microscope and oil immersion objective, original magnification 1000×.