Co-localization of multiple antigens and specific DNA. A novel method using methyl methacrylate-embedded semithin serial sections and catalyzed reporter deposition

Abstract

Co-localization of proteins and nucleic acid sequences by in situ hybridization and immunohistochemistry is frequently difficult as the process necessary to detect the target structure of one technique may negatively affect the target of the other. Morphological impairment may also limit the application of the two techniques on sensitive tissue. To overcome these problems we developed a method to perform in situ hybridization and immunohistochemistry on semithin sections of methyl methacrylate-embedded tissue. Microwave-stimulated antigen retrieval, signal amplification by catalyzed reporter deposition, and fluorescent dyes were used for both techniques, yielding high sensitivity and excellent morphological preservation compared to conventional paraffin sections. Co-localization of in situ hybridization and immunohistochemistry signals with high morphological resolution was achieved on single sections as well as on adjacent multiple serial sections, using computerized image processing. The latter allowed for the co-localization of multiple antigens and a specific DNA sequence at the same tissue level. The method was successfully applied to radiation bone marrow chimeric rats created by transplanting wild-type Lewis rat bone marrow into TK-tsa transgenic Lewis rats, in an attempt to trace and characterize TK-tsa transgenic cells. It also proved useful in the co-localization of multiple antigens in peripheral nerve biopsies.

Figure 1.

A–F: In situ hybridization of the TK-tsa transgene on MMA-embedded semithin sections of a TK-tsa transgenic Lewis rat. A–D: Representative serial 0.5-μm sections through the same nuclei reveal one or more in situ hybridization signals on each nucleus at least on one or on several of the sections. E and F: In situ hybridization performed on spleen sections of bone marrow chimeric rats. When wild-type Lewis rat bone marrow was transplanted into irradiated TK-tsa transgenic Lewis rats, the TK-tsa transgene was found only in resident endothelial cells (E, arrows). In contrast, when TK-tsa transgenic bone marrow was transplanted into irradiated wild-type Lewis rats, only bone marrow-derived round cells were positive for the transgene (F, arrows) whereas resident endothelial cells were negative. G–O: Immunohistochemistry and conventional staining on MMA-embedded tissue. G: ED1 antigen (green) on activated macrophage in a crushed peripheral nerve. H: Activated macrophages in crushed peripheral nerve visualized by the lectin GSI-B4 (green). I: Ramified microglial cell in brain (green, Iba1). J: Astrocyte visualized by an antibody against glial fibrillary acidic protein (red). K: Neurofilament visualized by antibody NR4 (green). L: Myelin basic protein delineating peripheral nerve myelin sheaths (green). M: Proliferating spleen cells as detected by an antibody against bromodeoxyuridine (green). Scale bar, 25 μm. N: Major histocompatibility complex class II (N, antibody Ox6; scale bar, 10 μm) in thymus (red). O: Toluidine blue routine staining of rat peripheral nerve. Scale bars, 5 μm (A–D), 10 μm (E, F, H, J, N), 20 μm (G, I, K, M), 30 μm (O), and 50 μm (L).

Figure 2.

A–D: Combined in situ hybridization and immunohistochemistry on the same semithin section of MMA-embedded crushed peripheral nerve of a TK-tsa transgenic rat. A: Tk-tsa in situ hybridization signal (red). B: immunohistochemistry using antibody ED1 to detect macrophages (green). There is no overlapping signal indicating complete absence of nonspecific binding subsequent to the double-labeling procedure. C: Phase contrast and 4,6-diamidino-2-phenylindol nuclear counterstain. D: Combined visualization of all signals revealed the presence of an ED1-positive macrophage carrying the TK-tsa transgene in its nucleus. E–H: Co-localization of three antigens by immunohistochemistry and TK-tsa DNA by in situ hybridization at the same tissue level using MMA-embedded adjacent serial semithin sections of crushed peripheral nerve of a TK-tsa transgenic rat. E: ED1 antibody labeling an activated macrophage (green). F: Myelin basic protein delineating myelin sheaths, some of them disintegrating. G: Bromodeoxyuridine labeling a proliferating cell. The perinuclear stain around positive cells is probably because of tyramide amplification. H: In situ hybridization for the Tk-tsa transgene (red). I–K: Documentation of myelin phagocytosis by macrophages because of the co-localization of myelin basic protein and the macrophage marker CD68 on serial sections of MMA-embedded human sural nerve. I: CD68 antibody KP1 labeling a rounded macrophage (green, arrows). J: Myelin basic protein inside the CD68 macrophage (green, arrows). K: Toluidine-blue routine staining of the myelin basic protein-positive macrophage (arrows). Scale bars, 7.5 μm (E–H), 10 μm (A–D), and 50 μm (I–K).