Effects of different fixatives on beta-galactosidase activity.

Abstract

W e read with great interest the article by Ma et al. (2002) on the effects of different fixatives on -galactosidase ( -Gal) activity. The authors evaluated the effects of four different fixatives on -Gal activity in kidneys from LacZ-stop-human alkaline phosphatase (Z/AP) double reporter mice. The authors demonstrated that 0.2% glutaraldehyde solution produced strong 5-bromo-4-chloro-3-indolyld -galactosidase (X-Gal) staining at room temperature (RT) for 4–8 hr. They also demonstrated that the X-Gal staining with 4% paraformaldehyde or 10% formalin fixative was markedly diminished at RT for 4–8 hr. Cell marking with reporter genes is useful in the study of cell lineage and differentiation in transplantation and regenerative research. Transgenic (Tg) mice overexpressing reporter genes, such as LacZ or green fluorescent protein (GFP), have been used for this purpose and have been valuable in the identification of cell migration and differentiation. We previously generated rats overexpressing enhanced GFP driven by a cytomegalovirus enhancer coupled to the chicken -actin promoter, and demonstrated the GFP-Tg rat as a useful tool for cell migration research after organ transplantation (Hakamata et al. 2001). We have also recently developed LacZ-Tg rats that abundantly express LacZ (unpublished). Herein we report a comparison of X-Gal staining under different fixative conditions in the skeletal muscles of LacZ-Tg rats. Samples of the skeletal muscles from LacZ-Tg rats were embedded in OCT compound (Miles Laboratory; Elkhart, IN), frozen in liquid nitrogen, and cut into thin (8–10m) sections. The sections were fixed with three different fixatives: (a) 0.2% glutaraldehyde; (b) 4% paraformaldehyde; and (c) 100% acetone. The periods of fixation were 10 min, 1 hr, 4 hr, or 8 hr at RT. The sections were washed three times in 0.1 M PBS (pH 7.4) for 5 min and then transferred to X-Gal staining solution [1 mg/ml of 5-bromo-4-chloro-