An Improved Method of Toluidine Blue Counterstaining of Neurons in Immunocytochemical Preparations

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Abstract
Immunocytochemical methods can identify individual neurons and processes made immunoreactive by virtue of the antigens they contain. However, frequently it is also useful to visualize surrounding nonimmunoreactive cells, but immunocytochemical procedures often interfere with the quality of subsequent counterstaining. This report describes an improved method of counterstaining immunocytochemical specimens with either aged (at least 1 year) or concentrated solutions of toluidine blue. This technique combines well with immunocytochemical preparations of at least two antigens, i.e., choline acetyltransferase and glutamic acid decarboxylase, to delineate nonimmunoreactive somata. Additionally, a method of photographing these color preparations is described that, by the use of an appropriate filter, allows one to illustrate sections essentially with and without blue counterstain in black and white photomicrographs.