Abstract
We describe a rapid method for the preparation and binding site labeling of cryostat sections for use in light microscopy. Instead of using antibodies to bind to specific sites, substance P, delta-sleep-inducing peptide, oxytocin, and dopamine were covalently attached to BSA and then the BSA-ligand complex was adsorbed on 5-nm colloidal gold particles. Bioassays carried out on isolated organs indicated that the physiological activity of the ligand GPL complex was maintained. Most of the technical steps included use of an ordinary microwave oven (MWO), with tissues exposed for less than 1 min in any given step. Cryostat sections of unfixed rat brain were pre-incubated for 50 sec in the MWO in a Tris-buffered solution (pH 7.4) containing 1.5% BSA, then further incubated for 50 sec in the MWO in Tris-buffered solution containing 1% gelatin and the diluted colloidal gold suspension. After washing, the preparations were postfixed for 30 sec in the MWO in 5% formaldehyde solution, pH 7.4. Finally, the cell-bound gold particles were enlarged by a silver-enhancing process and counterstained. Preparations observed at high magnification provided excellent resolution of the cell binding sites. Positive and negative controls performed by addition of BSA-conjugated ligands to the pre-incubation and incubation medium, and displacement of the markers by an excess of unbound ligand in the pre-incubation or the incubation medium, showed the specificity of the tissue labeling.