Combined intracellular injection of Neurobiotin and pre-embedding immunocytochemistry using silver-intensified gold probes in myenteric neurons

Abstract

We have developed methods to examine the neurochemistry of synaptic inputs to individual myenteric neurons labeled by dye injection through intracellular recording electrodes. Myenteric neurons of the guinea-pig ileum were filled with Neurobiotin, fixed, washed in 50% ethanol, exposed to sodium cyanoborohydride, incubated in avidin-biotin-horseradish peroxidase and incubated in antisera to calretinin or calbindin. The Neurobiotin-filled cells were revealed using the diaminobenzidine (DAB) reaction. The tissue was examined at the light microscope level to determine the morphology and projections of the Neurobiotin-filled neurons, and then incubated in 1 nm gold-labeled secondary antibodies. F..