Improved technique for detection of enhanced green fluorescent protein in transgenic mice

One of the most exciting recent advances in cell biology is the possibility to use the green fluorescent protein and its various mutated forms as repo rter proteins in studies carried out in vitro and in vivo. In the present s tudy, several detection techniques for the enhanced green fluorescent prote in (EGFP) were compared in transgenic mice, using fluorescence and confocal microscopy. In addition, different tissue preparation techniques (squash p reparations, vibratome sections, frozen sections) were evaluated. As a mode l we used transgenic mice expressing EGFP under the control of a 5.0-kb fra gment of the the glutathione peroxidase isoenzyme 5 protein promoter (GPX5- EGFP) or under a 3.8-kb fragment of the cysteine rich protein-1 promoter (C RISP1-EGFP). In the GPX5-EGFP mice, expression of EGFP was observed in the distal part of the caput epididymis, while the CRISP1 promoter directed EGF P expression in the tubular compartment of the testis. Among the various ti ssue preparation procedures tested, the best morphological and histological preservation, and reproducibility in EGFP detection, were obtained using f rozen sections after a slow tissue-freezing protocol developed in the prese nt study. After slow tissue freezing, specimens of testis and epididymis co uld be stored at -70 degreesC for at least six weeks without any affect on EGFP fluorescence. Hence, the method developed offers the possibility to an alyze EGFP fluorescence in tissues several weeks after specimen collection. The sensitivity achieved was equal to that found in immunohistochemistry, applying biotin-streptavidin-FITC detection. Confocal microscopy is known t o have the advantage that fluorescence cart be detected from cells in diffe rent layers. This was found to be important as regards detecting EGFP fluor escence because the fluorescence was destroyed at the cut surfaces of secti ons produced by either vibratome or cryomicrotome.