Richard W. Burry

Immunocytochemistry is a highly productive method in biomedical research used to identify proteins and other macromolecules in tissues and cells. Control samples are required to show label localization is correct, but the understanding and use of immunocytochemistry controls have been inconsistent. A new classification of immunocytochemical controls is proposed that will help in understanding this most important component of the experiment. The three types of controls required for immunocytochemistry are primary antibody controls that show the specificity of the primary antibody binding to the antigen, secondary antibody controls that show the label is specific to the primary antibody, and label controls that show the labeling is the result of the label added and not the result of endogenous labeling. Publications containing immunocytochemical results must give details of how these controls were performed.

Immunocytochemistry is used for antibody localization of proteins in cells and tissues. The specificity of the results depends on two independent criteria: the specificity of the antibody and of the method used. The antibody specificity is best determined by immunoblot and or immunoprecipitation. Absorption of the antibody with a protein does not determine that the antibody would have bound to the same protein in the tissue, and therefore is not a good control for antibody specificity. The specificity of the method is best determined by both a negative control, replacing the primary antibody with serum, and a positive control, using the antibody with cells known to contain the protein. With the increasing use of immunocytochemistry, it is important to be aware of the appropriate controls needed to show specificity of the labeling. (J Histochem Cytochem 48:163-165, 2000)

In pre-embedding EM immunocytochemistry with gold probes, the gold must be small enough to penetrate through cell membranes treated with mild detergents. Antibodies labeled with small gold probes (1-1.4 nm) are too small to be resolved in thin sections but can be seen if they are silver-enhanced after the gold has bound to the antigens in the cells. We investigated several aspects of gum arabic-silver lactate-hydroquinone enhancement solution (Danscher solution) by examining gold-conjugated antibodies embedded in agar, sectioned on a vibrotome, and enhanced with different solutions. The rate of silver enhancement was optimized in 50% gum arabic and 200 mM HEPES buffer, pH 5.8. We also examined chemicals used as developers and found that N-propyl gallate (NPG) gave a more uniform development than the routinely used hydroquinone (HQ). The diameter of the silver-enhanced particles after incubation in osmium tetratoxide (OSO4) decreased somewhat with longer incubation time and higher percentages, but the density (number per unit area) of silver-enhanced particles was little changed. The loss of silver-enhanced particle diameter was reduced by lowering the concentration of OSO4 to 0.1%. Comparison of commercial small gold probes showed that NPG enhancement of Nanogold gave more uniform particle size and a better correlation between enhancement time and particle density. When this procedure was applied to cell cultures with monoclonal antibodies, the silver-enhanced particles were similar to those in the agar sections. When free-floating tissue sections were used, longer silver enhancement times were needed to obtain similarly sized particles. This new NPG-silver-enhancement procedure offers a reliable and easy method to localize proteins in cultured cells and tissue sections by pre-embedding electron microscopic immunocytochemistry.

The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 μg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26(HER2/neu)) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ-deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4(+) or CD8(+) T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26(HER2/neu) tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs.