LacZ staining in paraffin-embedded tissue sections.

Abstract

Femora and tibiae of rats carrying leukemia from a L a dmarked acute promyelocytic leukemia-derived leukemic cell line (LT12NL15) were decalcified using EDTA and routinely embedded in paraffin. Sections IntroductionIn acute myelocytic leukemia (AML). infiltration of the bone marrow with leukemic cells is accompanied by severe suppression of normal hematopoiesis, eventually leading to life-threatening bleeding problems, recurrent infections, and anemia. To investigate whether this suppression of normal hematopoiesis is caused by spatial competition between normal hematopoietic stem cells and leukemic cells, Prim and Van Bekkum (1981) injected tritiated thymidine-labeled leukemic cells from the in vivo growing Brown Norway acute myelocytic leukemia (BNML) cell line, a wellcharacterized model of human AML (reviewed by Martens et al., 1990). into rats. It was subsequently observed that these cells were preferentially localized in the subendosteal region of the femoral bone marrow, in contrast to L4415 cells, a rat model for human acute lymphocytic leukemia (Kloosterman et al., 1992;Prins and Van Bekkum, 1981). This led to the assumption that AML cells specifically compete for space in the bone marrow compartment that is normally occupied by immature hemopoietic progenitor cells (Van Bekkum et al., 1981). To extend the study of the growth pattern and localization of acute myelocytic leukemia cells and to elucidate the role of adhesion molecules in this process, a more versatile and permanent method to mark leukemic cells was required.Genetic marking using the ErGherzchia coli B-galactosidase gene (LacZ) is a widely used method for the identification and localization of transplanted cells in vivo (Cui et al., 1994 an in vitro growing derivative of the BNML cell line (Lacaze et al., 1983). This resulted in the development of a genetically marked leukemic cell line, LT12NL15, which exhibits stable expression of large amounts of E. cofi P-galactosidase in the cytoplasm of every cell (Hendrikx et al., 1995;Yan et al., 1993). These cells were used to set up a sensitive system to study homing and growth of leukemic cells (Hendrikx et al., 1995). Here we report the development of an immunohistochemical staining method for Lac2 in paraffin sections of formalin-fixed, decalcified tibiae and femora of rats carrying LT12NL15 leukemia. This method allows detailed visualization of genetically marked leukemic cells within the undisturbed spatial context of the bone marrow and the enclosing bone. This procedure may also be of value to other studies in which detection of Lac2 labeled cells in p d i sections can o&r increased specificity, greater convenience, and superior tissue preservation compared to frozen sections. Materials and MethodsCells. LT12 is an in vitro as well as in vivo growing rat acute promyelocytic leukemia cell line, derived from the in vivo growing BNML cell line (Hagenbeek, 1977). LT12 cells were genetically marked using a retroviral vector containing the L a d gene, resulting in the subline LT12NL15. Briefl...

Visualizing the kinetics of tumor-cell clearance in living animals

“…Theoretically, other cell populations or biological functions that can be suitably labeled are amenable for study in living hosts with human diseases. Further modifications of this model system with closely related photoproteins that have similar biochemical characteristics but different emission wavelengths may permit the development of dual-color assays for monitoring tumor progression and interaction of the host response in the same animals (27).…”

Shifting foci of hematopoiesis during reconstitution from single stem cells

“…HSC function has been studied from a variety of experimental approaches, but HSC function typically must be assayed once tissues are removed from the body, often weeks or months after transplantation. Yet the critical early events of homing to the spleen and bone marrow (BM), seeding in the stromal microenvironment, and initial expansion of short-and long-term reconstituting cells occur within the first hours to days after transplantation (3)(4)(5)(6)(7)(8) and are therefore inaccessible to investigation in most studies. We have previously described an in vivo bioluminescence imaging (BLI) method to elucidate the spatiotemporal trafficking patterns of malignant cells, lymphocytes, and other mature immune cells within living animal models of human biology and disease (9)(10)(11)(12).…”

Techniques for Following Labeled Cells In Vivo: Use of X/Y FISH, Techniques to Optimize Fluorescent Detection, and Beta-Galactosidase Detection

“…Stable expression of beta-galactosidase expression within cells may be detected using a sensitive immunohistochemical system for detecting LacZ. The protocol for beta-galactosidase staining was modified from a published method by Hendrikx et al (9) Collect stomach tissue and wash well in 1× PBS.Pin tissue to a piece of dental wax placed in a well of a six well plate (see Note 4). Fix stomach tissue with 4% paraformaldehyde for 1 h on ice.…”