Parameters affecting imaging of the horseradish‐peroxidase‐diaminobenzidine reaction product in the confocal scanning laser microscope

Neurons intracellularly filled with biocytin and labelled with nickel‐intensified diaminobenzidine (DAB/Ni) can be imaged on the confocal scanning laser microscope in order to obtain three‐dimensional and optical section images of neurons. Intensification of the DAB reaction product with nickel was found to be crucial for obtaining a workable signal level. On the other hand, the high absorption of light by the reaction product severely attenuated the detection of structures lying directly underneath, and the intensity of the unattenuated laser used for imaging faded or damaged the DAB/Ni reaction product. We have determined that reduction of the laser intensity combined with the use of proper objective lenses and non‐laser‐based imaging for preliminary adjustments of the specimen all work to reduce or eliminate damage, and also improve the image. These items must be kept in mind when imaging and analysing DAB‐labelled structures in the laser‐based confocal microscope.