An improved method for beta-galactosidase activity detection on muscle tissue. A light and electron microscopic study.
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.
Gioglio
Luciana
L
Department of Experimental Medicine, Section of Human Normal Anatomy, Pavia, Italy.
Cusella
De Angelis M Gabriella
de AM
Boratto
Renata
R
Poggi
Paola
P
D000818:Animals; D004926:Escherichia coli / Q000201:enzymology; D017930:Genes, Reporter; D006651:Histocytochemistry; D051379:Mice; D008822:Mice, Transgenic; D008854:Microscopy, Electron; D018485:Muscle Fibers, Skeletal / Q000378:metabolism / Q000648:ultrastructure; D018482:Muscle, Skeletal / Q000166:cytology / Q000201:enzymology* / Q000648:ultrastructure; D018994:Myosin Light Chains / Q000235:genetics; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D018994:Myosin Light Chains; D001616:beta-Galactosidase