Optimization of differential immunogold-silver and peroxidase labeling with maintenance of ultrastructure in brain sections before plastic embedding

Abstract

The limited success of immunogold labeling for pre-embedding immunocytochemistry of neuronal antigens is largely attributed to poor penetration of large (5-20 nm) colloidal gold particles. We examined the applicability of using silver intensification of 1 nm colloidal gold particles non-covalently bound to goat anti-rabbit immunoglobulin (1) for single labeling of a rabbit antiserum against the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH), and (2) for immunogold localization of rabbit anti-TH simultaneously with immunoperoxidase labeling of a mouse monoclonal antibody against the opiate peptide, leucine-enkephalin (LE). Vibratome sections were collected from acrolein fixed brains of adult rats. These sections were immunolabeled without use of freeze-thawing or other methods that enhance penetration, but damage ultrastructure. By light microscopy, incubations in the silver intensifier (Intense M, Janssen) for less than 10 min at room temperature resulted in a brownish-red reaction product for TH. This product was virtually indistinguishable from that seen using diaminobenzidine reaction for detection of peroxidase immunoreactivity. Longer incubations produced intense black silver deposits that were more clearly distinguishable from the brown immunoperoxidase labeling. However, by light microscopy, the gold particles seen by electron microscopy were most readily distinguished from peroxidase reaction product with shorter silver intensification periods. The smaller size of gold particles with shorter periods of silver intensification also facilitated evaluation of labeling with respect to subcellular organelles. Detection of the silver product did not appear to be appreciably changed by duration of post-fixation in osmium tetroxide. In dual-labeled sections, perikarya and terminals exhibiting immunogold-silver labeling for TH were distinct from those containing immunoperoxidase labeling for LE. These results (1) define the conditions needed for optimal immunogold-silver labeling of antigens while maintaining the ultrastructural morphology in brain, and (2) establish the necessity for controlled silver intensification for light or electron microscopic differentiation of immunogold-silver and peroxidase reaction products and for optimal subcellular resolution.

Fig. 1

Light micrographs showing immunogold labeling for tyrosine hydroxylase (TH). A–D: photomicrographs of 4 coronal sections through the medial nucleus of the solitary tract that were sequentially incubated in rabbit TH-antiserum and goat anti-rabbit IgG bound to 1 nm gold particles. A and B were silver intensified for 8 min while C and D were for 10 min. A and C were post-fixed for 20 min and B and D for 90 min in 2% osmium tetroxide. There is no notable diminution of the immunogold labeling with longer postfixation periods. E–F: photomicrographs from sections that were dually labeled for TH using immunogold and for LE by the ABC method through the caudate nucleus. Incubations in the silver intensifier were 12 min in E and 14 min in F. The brown peroxidase reaction for LE in the perikarya (large arrows) appears distinct from the black immunosilver labeled axons (small arrows) in F. bv, blood vessel; ts, tractus solitarius; fb, fiber bundle. Bar = 50 μm.

Fig. 2

Ultrastructural localization of immunogold silver labeling for TH in a neuronal perikaryon in the medial nucleus of the solitary tract with 4 min silver intensification. The gold particles are exclusively localized within the cytoplasm of the neuron whose nucleus (nuc 1) is shown. The particles appear preferentially associated with the outer saccule of the rough endoplasmic reticulum (rer). The cytoplasm of another cell with nucleus (nuc 2) is unlabeled. The tissue was post-fixed 20 min in 2% OsO₄. Bar = 0.3 μm.

Fig. 3

Ultrastructural localization of immunogold silver labeling for TH in processes, presumably dendrites (den) in the medial nucleus of the solitary tract. Silver intensification for 2, 4 and 14 min in A–C, respectively. Small clusters of gold particles are barely detectable in A and assume larger and somewhat irregular shapes in B and C. The intensely labeled process to the right of the plate in C appears homogeneously electron dense resembling peroxidase immunoreactivity. Arrows in C indicate an even denser product resembling autoradiographic silver grains. A and B were from tissues incubated 20 min and C from tissue postfixed 60 min in 2% OsO₄. Bar = 0.4 μm throughout.

Fig. 4

Electron micrographs showing immunogold-silver labeling for TH in the medial nucleus of the solitary tract. A: after a 6 min silver intensification, individual, but irregular, silver clusters are seen in a longitudinally sectioned dendrite (den). This section was collected from the extreme outer surface of tissue postfixed 20 min in 2% OsO₄. B: after a 14 min silver intensification, the silver product has coalesced to resemble peroxidase (see Fig. 5) labeling. This section was postfixed for 60 min in 2% OsO₄ and was collected at a greater depth from the surface of the tissue than the section in A. ut = unlabeled axon terminal, arrows indicate regions of membrane for comparison of morphology obtained at different post-fixation times. Bars: A, 0.6 μm; B, 0.3 μm.

Fig. 5

Electron micrographs showing serial sections (A and B) immunolabeled with combined ABC for LE and gold-silver for TH. The tissue was postfixed 20 min in OsO₄. Peroxidase reaction product is localized in axon terminals containing LE. Silver particles indicative of TH-labeling also are seen in an axon terminal (TH). Silver enhancement time was 6 min at room temperature. Bar = 0.5 μm.