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A novel monoclonal antibody, RM-4, specifically recognizes rat macrophages and dendritic cells in formalin-fixed,
K Iyonaga1, M Takeya, T Yamamoto
1Second Department of Pathology, Kumamoto University School of Medicine, Japan.
Abstract:
An anti-rat macrophage/dendritic cell monoclonal antibody, RM-4, was produced using a homogenate of silica-induced lung granulomas of rat as immunogen. Immunohistochemistry demonstrated that RM-4 was specific for macrophage and dendritic cell populations residing in various organs and tissues. It did not react with any cells other than macrophage/dendritic cells. In the double staining of the spleen, RM-4-positive macrophages showed wider distribution than those of the four other anti-rat macrophage monoclonal antibodies compared. The immunoreactivity of RM-4 was well preserved not only in frozen sections but also in formalin-fixed, paraffin-embedded tissues. The isotype of the monoclonal antibody was IgG1 kappa and its antigen molecular weight was 46 kDa. Immunoelectron microscopy revealed positive reaction products for RM-4 on the membrane of endosomes and lysosomes in macrophages and epidermal Langerhans cells. Reaction intensity increased after thioglycolate elicitation or endocytosis regardless of ingested materials. From these data, it is concluded that RM-4 recognizes a membrane protein of endolysomes in macrophages and dendritic cells. The antigen may play a role in endolysosomal processing. RM-4 is considered to be a useful tool not only for identifying macrophage/dendritic cells both in frozen and paraffin-embedded tissues, but also for evaluating their endolysosomal processing.
Insights
A new monoclonal antibody, RM-4, identifies rat macrophages and dendritic cells in various tissues. This antibody targets an endolysosomal protein, aiding in cell identification and analysis of endolysosomal processing.
Area of Science:
- Immunology
- Cell Biology
Background:
- Macrophages and dendritic cells are critical immune cells involved in antigen presentation and immune regulation.
- Specific markers are needed to accurately identify and study these cell populations in various tissues.
Purpose of the Study:
- To develop and characterize a novel monoclonal antibody, RM-4, for the specific detection of rat macrophages and dendritic cells.
- To evaluate the utility of RM-4 in identifying these cells in different tissue types and preservation states.
- To investigate the cellular localization and potential function of the antigen recognized by RM-4.
Main Methods:
- Production of monoclonal antibody RM-4 using silica-induced lung granuloma homogenate.
- Immunohistochemistry and double staining for cell specificity and distribution analysis.
- Immunoelectron microscopy for ultrastructural localization of the antigen.
- Analysis of immunoreactivity in frozen and formalin-fixed, paraffin-embedded tissues.
Main Results:
- RM-4 specifically binds to rat macrophage and dendritic cell populations across various organs.
- RM-4 demonstrates broader distribution of macrophages in the spleen compared to other antibodies.
- The antibody retains immunoreactivity in both frozen and fixed tissues.
- RM-4 localizes to the membrane of endosomes and lysosomes, with increased intensity after elicitation or endocytosis.
- The recognized antigen has a molecular weight of 46 kDa and is an IgG1 kappa isotype.
Conclusions:
- RM-4 is a specific and versatile tool for identifying rat macrophages and dendritic cells in diverse tissue preparations.
- The antibody recognizes a membrane protein involved in endolysosomal processing within these cells.
- RM-4 can be utilized for both cell identification and functional assessment of macrophage/dendritic cell endolysosomal activity.