In the present study we describe a method for the histochemical demonstration of bacterial beta-D-galactosidase activity on skeletal muscle tissue processed for light and transmission electron microscopy. Hence allowing this enzyme to be accurately detected, bacterial beta-galactosidase expression was studied in transgenic mouse where the enzyme, with the nuclear localization signal (nlacZ), is under the transcriptional control of the striated muscle-specific promoter MLC3F. The chromogenic substrate, 5-bromo-3-indolyl-beta-D-galactopyranoside (Bluo-Gal), was used both to recognize labelled myofibers, and beta-gal positive organelles inside single myofibers. Moreover, because the preservation of enzyme is highly dependent on tissue fixation, we developed a suitable fixation solution allowing good preservation of both tissue and enzymatic activity. This was achieved by briefly fixing tissue (3 hours) in glutaraldehyde (2.5%) and paraformaldehyde (1%) in combination. This method should be taken into consideration when studying the gene therapy of muscle diseases because it is sensitive, inexpensive and not time consuming.

An improved method for ß-galactosidase activity detection on muscle tissue: a light and electron microscopic study / GIOGLIO L.; CUSELLA DE ANGELIS M. G.; BORATTO R.; POGGI P.;. - In: ANNALS OF ANATOMY. - ISSN 0940-9602. - STAMPA. - 184:2(2002), pp. 153-157.

An improved method for ß-galactosidase activity detection on muscle tissue: a light and electron microscopic study

GIOGLIO, LUCIANA;CUSELLA DE ANGELIS, MARIA GABRIELLA;BORATTO, RENATA;POGGI, PAOLA
2002

Abstract

In the present study we describe a method for the histochemical demonstration of bacterial beta-D-galactosidase activity on skeletal muscle tissue processed for light and transmission electron microscopy. Hence allowing this enzyme to be accurately detected, bacterial beta-galactosidase expression was studied in transgenic mouse where the enzyme, with the nuclear localization signal (nlacZ), is under the transcriptional control of the striated muscle-specific promoter MLC3F. The chromogenic substrate, 5-bromo-3-indolyl-beta-D-galactopyranoside (Bluo-Gal), was used both to recognize labelled myofibers, and beta-gal positive organelles inside single myofibers. Moreover, because the preservation of enzyme is highly dependent on tissue fixation, we developed a suitable fixation solution allowing good preservation of both tissue and enzymatic activity. This was achieved by briefly fixing tissue (3 hours) in glutaraldehyde (2.5%) and paraformaldehyde (1%) in combination. This method should be taken into consideration when studying the gene therapy of muscle diseases because it is sensitive, inexpensive and not time consuming.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11571/131483
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