alexa Optimal Immunofluorescent Staining for Human Factor IX
ISSN: 2157-7412

Journal of Genetic Syndromes & Gene Therapy
Open Access

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Research Article

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

Geoffrey L. Rogers and Brad E. Hoffman*

Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida, Gainesville, FL, 32610, USA

*Corresponding Author:
Brad E. Hoffman, Ph.D
University of Florida, College of Medicine
Department of Pediatrics
Division of Cellular & Molecular Therapy
Cancer & Genetics Research Complex
2033 Mowry Road Rm-207, Gainesville
FL 32610, USA
Tel: (352)273-8152 (O)
E-mail: [email protected]

Received date: July 12, 2012; Accepted date:August 13, 2012; Published date: August 15, 2012

Citation: Rogers GL, Hoffman BE (2012) Optimal Immunofluorescent Staining for Human Factor IX and Infiltrating T Cells following Gene Therapy for Hemophilia B. J Genet Syndr Gene Ther S1:012. doi:10.4172/2157-7412.S1-012

Copyright: © 2012 Rogers GL, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

 

Abstract

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.

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