A perfusion-fixation procedure for the concurrent demonstration of Timm's, horseradish peroxidase (HRP), and acethycholinesterase (AChE) histochemistry.

The sulfide-silver method of Timm has been a widely used histochemical technique to demonstrate the presence of heavy metals in biological tissue, particularly in the central nervous system. However, the use of this method or its several modifications results in less than optimal morphological preservation and requires embedding the tissue in paraffin or freezing it and cutting it directly onto slides with a cryostat. These procedures can decrease the sensitivity and limit the application of other histochemical procedures, particularly when experiments necessitate processing large specimens or reaction procedures require techniques using free-floating sections. A perfusion-fixation protocol is described that yields sufficient fixation to cut whole frozen blocks of tissue with a sliding microtome, permits the use of free-floating sections, and allows the concurrent demonstration of horseradish peroxidase and acetylcholinesterase histochemistry without loss of sensitivity. The method consists of a short initial exposure to a sodium sulfide solution followed by a prolonged exposure to a combined sulfide-aldehyde fixative solution.