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.

Conditional deletion of the NMDA-NR1 receptor subunit gene in the central nucleus of the amygdala inhibits naloxone-induced conditioned place aversion in morphine-dependent mice

“…Mouse brains were prepared for electron microscopy as previously described (Leranth and Pickel, 1989;Chan et al, 1990). Briefly, mice were deeply anesthetized with sodium pentobarbital (150 mg/kg, i.p.)…”

N-methyl-d-aspartate receptor independent changes in expression of polysialic acid-neural cell adhesion molecule despite blockade of homosynaptic long-term potentiation and heterosynaptic long-term depression in the awake freely behaving rat dentate gyrus

“…No immunostaining was seen on any of these sections. For EM pre-embedding immunogold-silver labelling (Chan et al, 1990), sections were rinsed in 0.01 M phosphatebuffered saline pH 7.4 (PBS), and blocked in 0.8% BSA and 0.1% coldwater fish gelatin in PBS (BSA/gelatin) for 10 min and incubated in the primary antibody at a dilution of 1:50 under the same conditions as for light immunocytochemistry. Following this incubation, sections were processed for 2 h in a 1:50 dilution of rabbit anti-goat IgM conjugated with 1 nm colloidal gold (British Biocell International, Cardiff, UK) in BSA/gelatin, and then rinsed in BSA/gelatin followed by PBS.…”

Changes in the subcellular distribution of NADPH oxidase subunit p47phox in dendrites of rat dorsomedial nucleus tractus solitarius neurons in response to chronic administration of hypertensive agents

“…There is the possibility that ABC/DAB reaction product will be intensified by silver enhancement. In our dual labeling experiments, the primary antisera labeled with a gold secondary was omitted as a control for this possibility (8). It should also be pointed out that peroxidase reaction product is typically not particulate, but somewhat diffuse in nature, and thus with careful visual inspection not easily confused with silverenhanced immunogold.…”

“…Immunogold electron microscopic cytochemistry has the spatial resolution necessary to detect protein immunolabeling at distinct subcellular localizations, and is also compatible with dual immunoperoxidase labeling for related antigens (8,27). We used immunogold electron microscopy to characterize the subcellular distributions of the p47 phox subunit in different size dendritic processes in response to chronic subcutaneous infusion of AngII.…”