Figure 1. Flow diagram of essential processing steps prior to the immunostaining procedure. For explanation see text.

Figure 2. Schematic representation of the most common immunohistochemical staining procedures. The steps for each procedure are numbered. For explanation see text and Table 1.

Figure 3. Immunoperoxidase staining of a rat spleen section. An acetone-fixed frozen section from rat spleen was incubated first with a mouse monoclonal antibody directed to B cells (HIS14) and followed by a horseradish peroxidase-labelled secondary antibody directed against mouse antibodies. The peroxidase label was revealed using diaminobenzidine, resulting in a brown precipitated reaction product at the site of binding of the monoclonal antibody to the cells. B cells in this section are stained brown and are located in the typical B cell areas: follicles (F) and marginal zone (MZ). T lymphocytes are located in the periarteriolar lymphocyte sheath (PALS) and are unstained.

Figure 4. Two-colour immunohistochemical staining of heart tissue after transplantation. To identify infiltrating T and B cells in cardiac tissue undergoing chronic rejection in rats, an indirect double-staining technique was performed. Fixed cryostat section of tranplanted rat heart was incubated with a mixture of an IgG1 mouse monoclonal antibody directed against rat T cells (OX19) and an IgG2b mouse monoclonal antibody directed against rat B cells (HIS24). These antibodies were revealed with alkaline phosphatase-conjugated goat anti-mouse IgG1 and horseradish peroxidase-conjugated goat anti-mouse IgG2b, respectively. In this section the infiltrating T cells are stained blue and B cells are stained red. Courtesy of J. L. Hillebrands, University of Groningen.

Figure 5. Immunoperoxidase and immunofluorescence staining of human skin. Frozen sections of human skin were stained for laminin 5 using a mouse monoclonal antibody to laminin 5, followed by polyclonal rabbit anti-mouse antibodies, conjugated to horseradish peroxidase (A), or to FITC (B). Peroxidase was revealed using diaminobenzidine (brown) and nuclei were stained with haematoxylin (blue). Laminin 5 is expressed by the epidermal basement membrane zone (arrow). Ep = epidermis. Courtesy of M. van der Deen and Dr. M. C. J. M. de Jong, University of Groningen.

Figure 6. Immunofluorescence staining of human skin. Human skin sections were stained by immunofluorescence as described for Figure 4, using monoclonal antibodies directed to bullous pemphigoid antigen BP230 (A) or BP180ic (B). Nuclei are stained blue using bisbenzimide. BP230 is expressed by the epidermal basement membrane zone (arrow) and BP180ic also by the lamina basalis of the epidermis (Ep). Courtesy of M. van der Deen and Dr. M. C. J. M. de Jong, University of Groningen.

Figure 7. Three-colour immunofluorescence staining of mouse lymphocytes after BrdU incorporation. These photographs show two peritoneal B cells (B-1 cells) of a mouse fed with BrdU in the drinking water. Peritoneal cavity cells were stained with a fluoresceinated (FITC, green fluorescence) rat monoclonal antibody directed CD5 (B) and a rhodamine (TRITC, red fluorescence) conjugated rat monoclonal antibody to mouse IgM (C). After cytocentrifugation, fixation and denaturation of the cells, BrdU incorporation was visualized with an IgG1 mouse monoclonal antibody to BrdU, followed by biotinylated goat anti-mouse IgG1 and finally AMCA (blue fluorescence)-conjugated avidin (D). The pictures clearly show that both cells express CD5 and IgM, whereas only one of them had incorporated BrdU and thus had gone through a cell cycle during the period of BrdU feeding. (A) Phase contrast picture of the two peritoneal cells. Reproduced from Deenen GJ and Kroese FGM (1993) European Journal of Immunology 15: 12–16; with permission.

Figure 8. Confocal scanning laser microscopy image of six optical planes of a hepatocyte couplet. Some hepatocytes isolated from a (rat) liver stay attached to each other, forming a bile canaliculus (apical domain) in culture. Apical trafficking of the multidrug resistance protein 2 (MRP2), a glutathione S-conjugate transporter, was studied in hepatocyte couplets. The image shows six optical planes through a hepatocyte couplet that was treated with an inhibitor of microtubule polymerization (nocodazole) and then stained for MRP2 with a polyclonal antibody followed by a FITC-labelled secondary antibody. The upper left image shows the top of the couplet and the lower right image shows the bottom (the side that is attached to the culture dish). MRP2 is predominantly localized in a vesicular compartment just below the plasma membrane, indicating that apical trafficking of MRP2 is dependent on microtubules. Courtesy of Dr. J. Roelofsen, University of Groningen.

Figure 9. Immunoelectronmicroscopy of brain tissue using immunogold. Ultrathin sections of rabbit brain were stained for the presence of the most frequently occurring inhibitory neurotransmitter GABA using a postembedding, indirect immunogold method. Sections were incubated with polyclonal rabbit anti-GABA antibody, followed by secondary antibodies directed against rabbit antibodies conjugated with 15 nm gold particles. The gold particles, and thus GABA, are predominantly observed in axonal terminal (AT) in contact with a proximal dendrite (D) of a neuron in the nucleus of the optic tract in the rabbit. Note that mitochondria (arrow) within the labelled terminal also exhibit significant amounts of GABA. Courtesy of Dr. J. J. L. van der Want, University of Groningen.