Immunohistochemical Markers for the Rodent Immune System

Department of Molecular Cell Biology and Immunology, VU University Amsterdam, Amsterdamo, North Holland, Netherlands
Toxicologic Pathology (Impact Factor: 2.14). 02/2006; 34(5):616-30. DOI: 10.1080/01926230600941340
Source: PubMed


The responses to insults including chemical toxins, irradiation and infectious agents involve morphologic, biochemical and molecular changes in the immune system. The changes in specific tissues and cells often can be detected by histopathology and its associated field of immunohistochemistry (IHC). Cells normally express specific proteins (antigens) that can be detected by IHC. When responses to xenobiotics occur, cells often up or down regulate proteins. The art of IHC requires specialized procedures for detection of antigens. Fixation, tissue processing, immunoreactions and antigen retrieval methods are important elements of IHC. We review the antibodies, their sources, use of frozen or fixed paraffin-embedded tissues and specific IHC methods including antigen retrieval and illustrate how they can be effectively used to characterize the immunotoxicologic effects of agents.

Full-text preview

Available from:
  • Source
    • "The primary antibodies were labeled with FITC-conjugated goat anti-rabbit IgG [H + L] (green, ZSGB-BIO) and TRITC-conjugated goat anti-mouse IgG [H + L] (red, Boster), Dylight™594 goat anti-Rabbit IgG [H + L], and Dylight™488 goat anti-Mouse IgG [H + L] (Muletiscience). The protocols were performed as in previous articles [18–20]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background. The interactions between cancer-associated fibroblasts (CAFs) and cancer cells or tumor-infiltrating lymphocytes (TILs) and cancer cells play important roles in cancer progression and metastasis. However, studies related to the crosstalk between CAFs and TILs in tumor microenvironment (TME) are still lacking. In this study, we mainly investigated the interactions between CAFs and TILs. Material and methods. The distribution of TILs rich in regulatory T cells (Tregs) in breast cancer tissues was evaluated using hematoxylin-eosin staining and immunohistochemistry with anti-CD3, anti-Foxp3, and anti-α-smooth muscle actin antibodies. Homologous CAFs/normal fibroblasts (NFs) and TILs cultured in vitro were identified and detected using immunocytochemistry and flow cytometry (FCM). The direct interaction among these cell types was studied via a factorial design in a co-cultured system. Their indirect interaction was assayed using Transwell plates. The cell cycle and apoptosis of CAFs/NFs co-cultured with TILs was analyzed using propidium iodide staining. Results. Histochemistry demonstrated most of the TILs including Tregs, were distributed in the cancer stroma, adjoining to CAFs. This finding implies that both cell types interact closely in the TME. Identification of the cultured cells showed that CAFs maintained their activated phenotype within limited passages in vitro, and that the TILs population contained a high percentage of Tregs. Data analysis of the factorial design suggests significant interactions among CAFs, NFs, and TILs in both direct and indirect contact ways. The CAFs and NFs were suppressed signally by TILs, which are probably induced by the secretory cytokines derived from TILs or Tregs. Although apoptosis was not detected in CAFs/NFs, the cell cycle assay suggested that the CAFs/NFs were arrested in the G2/M phase by the TILs and their secretory cytokines. Conclusion. CAFs and NFs were dramatically suppressed by Tregs-rich TILs. This suggests the interaction between TILs and CAFs might modify the TME in an unknown manner.
    Full-text · Article · Jan 2013 · Acta oncologica (Stockholm, Sweden)
  • Source
    • "Some parts of lymph node specimens prepared by perfusion-fixation were infiltrated in PBS containing 30% sucrose [44], and 6 µm cryosections were cut. They were immunostained for LYVE-1 with the common immunoperoxidase-DAB method as reported before [40, 41]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The "in vivo cryotechnique" (IVCT) is a powerful tool to directly freeze living animal organs in order to maintain biological components in frozen tissues, reflecting their native states. In this study, mesenteric lymph nodes of living mice were directly frozen with IVCT, and we did morphological studies and immunohistochemical analyses on a hyaluronic acid receptor, LYVE-1. In lymph nodes, widely open lymphatic sinuses were observed, and many lymphocytes adhered to inner endothelial cells along subcapsular sinuses. The LYVE-1 was clearly immunolocalized at inner endothelial cells of subcapsular sinuses, as well as those of medullary sinuses. Conventional pre-embedding electron microscopy also showed LYVE-1 immunolocalization along both the apical and basal sides of cell membranes of inner endothelial cells. By triple-immunostaining for LYVE-1, smooth muscle actin, and type IV collagen, the LYVE-1 was immunolocalized only in the inner endothelial cells, but not in outer ones which were surrounded by collagen matrix and smooth muscle cells. Thus, the functional morphology of lymph nodes in vivo was demonstrated and LYVE-1 immunolocalization in inner endothelial cells of subcapsular sinuses suggests hyaluronic acid incorporation into lymph node parenchyma.
    Full-text · Article · Apr 2011 · ACTA HISTOCHEMICA ET CYTOCHEMICA
  • Source
    • "Mice were observed for 8 to 12 months and necropsied when signs of splenomegaly, lymphadenopathy, labored breathing or lethargy were noted or the experiment was terminated at 12 months. Diagnosis was based on gross findings, microscopic examination of H&E stained formalin fixed, paraffin embedded tissues or studied by IHC using the anti-p30 antibody, anti-CD3 for T-cell lineage identification (DAKO Corporation, Carpinteria, CA Catalog # A452), and anti-PAX5 for B-cell lineage (Goat anti-Pax 5, Santa Cruz Biotechnology, Santa Cruz, CA, Catalog #sc-1974) [14]. Criteria for histopathological diagnosis were as described [15]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The mouse macrophage-like cell line RAW264.7, the most commonly used mouse macrophage cell line in medical research, was originally reported to be free of replication-competent murine leukemia virus (MuLV) despite its origin in a tumor induced by Abelson MuLV containing Moloney MuLV as helper virus. As currently available, however, we find that it produces significant levels of ecotropic MuLV with the biologic features of the Moloney isolate and also MuLV of the polytropic or MCF class. Newborn mice developed lymphoma following inoculation with the MuLV mixture expressed by these cells. These findings should be considered in interpretation of increasingly widespread use of these cells for propagation of other viruses, studies of biological responses to virus infection and use in RNA interference and cell signalling studies.
    Full-text · Article · Feb 2008 · Retrovirology
Show more