Wheat germ agglutinin-apoHRP gold: a new retrograde tracer for light- and electron-microscopic single- and double-label studies

Abstract

In this report, we describe a new colloidal-gold-labelled retrograde tracer, wheat germ agglutinin (WGA) conjugated to enzymatically inactive horseradish peroxidase (apoHRP). This protein gold complex (WGAapoHRP-Au) is a sensitive marker for retrograde tracing of the projections of CNS neurons at the light-microscopic (LM) level when a silver-enhancement procedure is used to detect the gold in the tracer. For electron-microscopic (EM) analysis, the silver-enhanced sections undergo a further gold-toning step. This protects against rapid oxidation and dissolution of the silver precipitate during the osmication procedure. A major advantage of WGAapoHRP-Au is that it can be used in a variety of multiple-labelling studies. When the retrograde transport of the new tracer is combined with that of the fluorescent dye, True Blue, neurons that have bifurcating axons can be readily demonstrated. Simultaneous immunofluorescent detection of the cytochemistry of the double-retrogradely labelled neurons is also possible. In contrast to a WGA-HRP gold complex, the new complex has no enzymatic activity. Thus HRP-based techniques (e.g., anterograde transport of WGA-HRP or peroxidase-antiperoxidase immunocytochemistry) can be performed on tissue that contains retrogradely labelled neurons marked with WGAapoHRP-Au without having to pretreat tissue so as to destroy endogenous HRP enzyme activity. At the EM level, the gold is readily distinguished from DAB immunoreaction product. This makes both LM and EM double-labelling studies possible. The great sensitivity of the new tracer, its compatibility with a variety of aldehyde fixatives, its ease of detection, and the fact that it can be simultaneously used with several fluorescent and HRP-based immunocytochemical and tracing techniques make WGAapoHRP-Au a valuable tool for LM and EM characterization of CNS cytochemistry and connectivity.