Signal Amplification in Immunohistochemistry at the Light Microscopic Level Using Biotinylated Tyramide and Nanogold-Silver Staining

Abstract

(CJFVN) S U M M A R Y Signal amplification techniques greatly enhance the sensitivity of immunohistochemical (IHC) and in situ hybridization (ISH) methods. In particular, catalyzed signal amplification (CSA) using labeled tyramide or Nanogold-silver staining is an important signal amplification tool. We have applied a combination of both techniques, as has been introduced for ISH, for a further increase in sensitivity of an IHC method to detect cathepsin B. This lysosomal proteinase can also be expressed extracellularly, particularly in relation to cancer metastasis. Higher sensitivity of the IHC method was needed because existing methods failed to demonstrate cathepsin B protein where cathepsin B activity was found with a fluorescence enzyme histochemical method. Combined CSA and Nanogold-silver staining provided the sensitivity that was required. Moreover, this signal amplification method enabled the use of a 10-fold lower concentration of primary antibody (1 g/ml). Nonspecific background staining was low provided that endogenous biotin, avidin, and peroxidase were completely blocked. The method was reproducible when all steps, and particularly the silver enhancement step, were rigidly controlled. The method resulted in localization patterns of cathepsin B protein that were in agreement with those of cathepsin B activity in serial sections of rat liver containing colon cancer metastases. We concluded that combined application of CSA and Nanogold-silver staining provides high sensitivity for immunohistochemical methods and that activity localization by an enzyme histochemical method is a very attractive alternative to IHC localization of an enzyme because it is at least as sensitive, it is rapid and simple, and it provides direct information on the function of an enzyme.

Immunophenotyping of HPV Types 16 and 18 among Sudanese Patients with Oral Lesions

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Cellular localization of lead using an antibody‐based detection system and enzyme activity changes in the gills and digestive gland of the blue mussel Mytilus edulis

“…For ultrastructural localization of the heavy metal Pb, we applied the preembedding technique and a signal amplification system based on catalyzed signal amplification according to Koehler et al [14] and gold‐substituted silver‐intensified peroxidase according to Van den Pol and Gorcs [15]. Tissues were sectioned with a tissue chopper (Linton Instrumentation, Norfolk, UK) into 0.5 × 0.5 mm pieces and were fixed in 2% paraformaldehyde and 0.1% glutaraldehyde for 2 h at 4°C and in 2% paraformaldehyde over night.…”

Imaging Enzymes at Work: Metabolic Mapping by Enzyme Histochemistry

“…Ideally, the substrate reaches the same concentration in cells as in the incubation medium in a short period of time, whereas the product remains in cells. In that way, enzyme reactions can be monitored (A) Immunohistochemical localization of cathepsin B protein using an immunogold method and silver enhancement (Koehler et al 2000). Cathepsin B protein is represented by white dots visualized by reflection contrast microscopy.…”