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Published on: November 14, 2016
Immunohistochemical analysis of rat liver using a monoclonal antibody (HAM8) against gap junction
1Department of Anatomy, Yamaguchi University School of Medicine, Japan.
Insights
A new antibody, HAM8, identifies connexin 32, a key gap junction protein in rat liver. This protein
Area of Science:
- Cell Biology
- Developmental Biology
- Immunology
Background:
- Gap junctions are crucial for intercellular communication.
- Understanding connexin distribution is vital for developmental and disease studies.
- Connexin 32 (Cx32) is a major gap junction protein in the liver.
Purpose of the Study:
- To develop monoclonal antibodies against rat liver gap junctions.
- To investigate the developmental expression of gap junctions.
- To analyze gap junction polarity in hepatocytes in vitro and in vivo.
Main Methods:
- Production of monoclonal antibodies against rat liver gap junction fractions.
- Immunofluorescence staining on frozen sections of various rat tissues and species.
- Analysis of primary cultured hepatocytes and rat hepatoma cells.
- Intravenous injection of HAM8 IgG antibody in rats.
Main Results:
- HAM8 antibody specifically recognizes rat connexin 32 (27-kDa).
- HAM8 antigen is found in hepatocytes, exocrine pancreas, and salivary gland, but not in kidney, heart, esophagus, or thymus.
- HAM8 antigen expression increases with fetal liver development and is present in adult liver.
- In cultured hepatocytes, HAM8 antigen is intercellular but diminishes on free surfaces within 4 hours.
- HAM8 antigen is absent in AH-7974 rat hepatoma cells.
- Intravenous injection of HAM8 IgG leads to signal expression in the liver.
Conclusions:
- HAM8 antibody is a specific tool for detecting rat connexin 32.
- Connexin 32 expression is developmentally regulated in the rat liver.
- Hepatocyte gap junctions exhibit polarity, with rapid turnover or redistribution in culture.
- Connexin 32 is not expressed in AH-7974 hepatoma cells, suggesting potential roles in tumorigenesis.
Abstract:
Four monoclonal antibodies were raised against crude gap junction fractions of rat liver to clarify the distribution of gap junctions during animal development and to analyze gap junction expression in vivo and the polarity of hepatocytes in vitro. Among the monoclonal antibodies obtained, HAM8 antibody recognized the 27-kDa rat liver gap junction protein connexin 32. This antibody recognized gap junctions at the contiguous faces of hepatocytes, and the antigen was also observed in exocrine pancreas and salivary gland but not in kidney, heart, esophagus, or thymus. HAM8 did not react with amphibian or fish liver, heart, esophagus, stomach, or intestine as assessed via the immunofluorescence method on frozen sections. A few hepatocytes and many hemopoietic cells were seen in rat fetal liver at 15 days of gestation. HAM8 antigen was expressed on some hepatocytes but not on any hemopoietic cells. As the fetus grew, the number of hepatocytes in the liver increased gradually, together with the amount of HAM8 antigen. The distribution of HAM8 antigen at 25 days after birth was similar to that in adult liver. When the expression of HAM8 antigen was examined in primary cultured hepatocytes using the immunofluorescence method, the antigen was observed clearly between the hepatocytes. However, most of the HAM8 antigen on the free surface of hepatocytes disappeared within 4 hr. HAM8 antigen was not expressed on AH-7974 rat hepatoma cells when they formed small islets in the rat peritoneal cavity or within the liver. When HAM8 IgG antibody was injected intravenously, the HAM8 signal was expressed in the liver.
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