With a view to improving the immunohistochemical technique for the demonstration of bioactive substances, i.e., neuropeptides and biogenic amines in the mammalian central nervous system, the procedures of immunoperoxidase methods were examined and some modifications for obtaining consistent results were developed.
Brains of pharmacologically untreated animals were utilized as the material of the present study. The time through thoracotomy and preperfusion was reduced as much as possible. Perfusion fixation was performed at an increased rate, using a blood pump (hemolizer). The first fixative was a mixture of 4% formaldehyde, 0.2% picric acid, and 0.5% glutaraldehyde in phosphate buffer; the second was 4% formaldehyde and 0.2% picric acid in phosphate buffer acidified to pH6.5 with acetic acid; and the third was buffered formaldehyde solution. The osmotic pressure of all these fixatives was 1550-1650mOsM. Sections 25μm thick were produced on a Microslicer, followed by application of the freezing-thawing technique. The free-floating sections were incubated in a low concentration of antibody solution diluted by 0.5% Triton X-100 in phosphate buffer for a longer period than usual, under cool condition. The reactive substances resulting from the avidinbiotin-peroxidase complex method were enhanced by osmication. With these methods, the serotonin and GRH neurons could be clearly and finely visualized.