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Effects of Different Fixatives on ß-galactosidase Activity

Correspondence to: Masafumi Takahashi, Div. of Organ Replacement Research, Center for Molecular Medicine, Jichi Medical School Minamikawachi-machi, Tochigi 329-0498, Japan. E-mail: masafumi@jichi.ac.jp

We read with great interest the article by Ma et al. 2002 on the effects of different fixatives on ß-galactosidase (ß-Gal) activity. The authors evaluated the effects of four different fixatives on ß-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-indolyl-ß-D-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 ß-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 We have also recently developed LacZ-Tg rats that abundantly express LacZ (unpublished).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 (8–10-µm) 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-3-indolyl-ß-d-galactopyranoside, 2 mM MgCl₂, 5 mM potassium hexacyanoferrate (III), 5 mM potassium hexacyanoferrate (II) trihydrate] at 37C for 30 min. Consistent with the findings by Ma et al. 2002 , 0.2% glutaraldehyde showed strong X-Gal staining at RT for 4 hr, and a slightly diminished intensity after 8 hr (Fig 1A). However, fixation with 4% paraformaldehyde showed no X-Gal staining at RT at any time point (Fig 1B). The differences between our findings and those of Ma et al. 2002 might be due to differences in the fixation process (i.e., fixation was performed before or after tissues were frozen). Importantly, 100% acetone produced stronger X-Gal staining than 0.2% glutaraldehyde (Fig 1C). These results suggest that 100% acetone was effective as fixative for a period of less than 8 hr, even at RT. These observations provide useful information for the evaluation of LacZ expression in transplantation and regenerative research.

View larger version (52K): [in this window] [in a new window]   Figure 1. Skeletal muscle sections from LacZ-Tg rats were fixed with 0.2% glutaraldehyde (A), 4% paraformaldehyde (B), and 100% acetone (C) at RT for 8 hr, and stained with X-Gal solution at 37C for 30 min. Bar = 50 µm.

Literature Cited

Hakamata Y, Tahara K, Uchida H, Sakuma Y, Nakamura M, Kume A, Murakami T et al. (2001) Green fluorescent protein-transgenic rat: a tool for organ transplantation research. Biochem Biophys Res Commun 31:779-785

Ma W, Rogers K, Zbar B, Schmidt L (2002) Different fixatives on ß-galactosidase activity. J Histochem Cytochem 50:1421-1424[Abstract/Free Full Text]

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