Focus: Anti-β-Galactosidase Antibody for Immunohistochemistry

Use of anti-β-galactosidase antibody to identify transplanted bone marrow-derived cells expressing bacterial β-galactosidase

The Anti-β-Galactosidase, Purified Monoclonal Antibody (Cat.# Z3781, Z3783), was used to identify transplanted bone marrow cells, which expressed bacterial β-galactosidase, and were recruited to gastric cancer sites. The Anti-β-Galactosidase mAb was able to distinguish the bacterial enzyme from the endogenous mammalian enzyme.

From the article: Houghton, J. et al. (2004) Science 306, 1568–71. Department of Medicine and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA.

Published in March 2006

Introduction

Bone marrow–derived cells (BMDCs) are frequently recruited to sites of tissue injury and inflammation and may be a potential source of malignancy. The possible role of homing and engraftment of BMDCs in gastric cancer associated with chronic Helicobacter infection was studied by the authors of this paper. The established
H. felis C57BL/6 mouse model of gastric cancer was used with transplanted ROSA26 (C57BL/6JGtrosa26) BMDCs, which expressed the transgenic marker bacterial β-galactosidase so that the transplanted cells could be distinguished from normal stomach cells. Immunohistochemistry (IHC) was performed using the Promega Anti-β-Galactosidase, Purified Monoclonal Antibody (Cat.# Z3781, Z3783) to identify cells expressing bacterial β-galactosidase. This antibody showed no cross-reactivity to antigenically similar mammalian enzymes.

Materials and Methods

Bone marrow was harvested from 6–8 week old ROSA26 mice and transplanted into irradiated recipient C57BL/6J mice. The controls were non-transplanted C57BL/6J mice and C57BL/6J mice transplanted with C57BL/6J marrow. The experiment was initiated by infecting the ROSA26-transplant mice and control mice with 1 × 10⁷ colony forming units of Heliobacter felis strain 49179 every other day for three days. After 52 weeks, the mice were euthanized, the gastric tumor tissue removed and 4µm fixed sections prepared for dual fluorescence IHC to confirm the presence of transplanted BMDC in the gastric tumors.

Samples were sequentially labeled, first for the epithelial cell marker cytokeratin (AE1/AE3, DakoCytomation), and then for bacterial β-galactosidase using the Anti-β-Galactosidase, Purified Monoclonal Antibody (Promega). The ARK™ kit (Dako) was used for this mouse-on-mouse IHC application. Antibody binding was demonstrated by indirect fluorescent labeling with streptavidin (SA)-fluorescein for cytokeratin and SA-Cy^®3 for β-galactosidase. Slides were mounted and nuclei counterstained using VECTASHIELD^® + DAPI (Vector Laboratories). The Promega antibody was chosen because a previously evaluated rabbit polyclonal anti-β-galactosidase antibody failed to distinguish between transplanted BMDCs from ROSA26 transgenic mice and host cells bearing closely related endogenous murine enzymes.

 

Figure 1. Immunohistochemistry of murine high-grade gastrointestinal intraepithelial neoplasia (GIN) after transplantion of ROSA26 bone marrow and H. felis infection. Glands within the GIN identified in the mouse model were subjected to dual fluorescence IHC using the Anti-β-Galactosidase mAb and a pan-cytokeratin antibody. The colocalization of these two fluorophors (red + green = yellow) confirms the integration and differentiation of BMDCs into gastric epithelium. Image used with the kind permission of J. Houghton, University of Massachusetts Medical School Worchester, A. Rogers, Massachusetts Institute of Technology, and Science Magazine. Copyright 2004 AAAS.

Results and Discussion

The double fluorescent label experiment with these two antibodies (anti-β-galactosidase = red Cy^®3, cytokeratin = green fluorescein; Figure 1) demonstrated that BMDCs recruited to the stomach in response to chronic inflammation differentiated into the gastric epithelial phenotype and also underwent neoplastic transformation, presumably as a result of their plasticity and exposure to an altered milieu of growth signals and cytokines. The use of the Anti-β-Galactosidase mAb enabled the specific identification of the transplanted BMDCs. The authors concluded that pluripotent bone marrow-derived cells may give rise to epithelial neoplasms with potential implications for stem cell therapies and approaches to the treatment of cancer.