Optimal Immunofluorescent Staining for Human Factor IX and Infiltrating T Cells following Gene Therapy for Hemophilia B.

Immunofluorescent imaging is a valuable tool for investigating the outcome of gene therapy within the transduced tissue. With a multi-labeling technique, it is possible to both characterize local expression of the transgene and to evaluate the severity of the adaptive immune response through cytotoxic T cell infiltration. It is critical that the experimental parameters are optimal in order to prevent misinterpretation of important pathological events. To optimize this staining protocol, murine liver and skeletal muscle was transduced using recombinant adeno-associated virus encoding human factor IX. After testing several common cryo-preservative and fixative techniques, we found that optimal tissue integrity and antigen (factor IX and CD8) detection was achieved by freezing muscle tissue on liquid nitrogen-cooled isopentane (also called methylbutane or 2-methylbutane), followed by fixation with acetone at room temperature. The staining protocol described herein requires only about two hours, yet maintains exquisite specificity even at high magnification under confocal microscopy.