Abstract
One of the first applications of confocal microscopy was reflection imaging of unstained neurons. However, most applications employ either Golgi (silver impregnation) or peroxidase staining. Golgi preparations have been imaged by tandem scanning, but both need optimization for confocal laser scanned microscopy (CLSM) due to low signal levels and internal reflections. The imaging wavelength is critical. Specimen absorption of the argon ion laser lines is an order of magnitude larger than the HeNe red line. We compare the imaging of Golgi and peroxidase stained neurons using both lasers.Rat hippocampal neurons were filled with biocytin, fixed in 4% paraformaldehyde, incubated in avidin-HRP with Triton and reacted with DAB with or without Ni. Specimens were imaged in permount using a Bio-Rad MRC 500 and Olympus BH-2.
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