Use of Contrasting Fluorescent Dye as Counterstain in Fixed Tissue Preparations

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Abstract
A major problem in fluorescent antibody studies is the nonspecific uptake of the fluorescein dye in fixed tissues. A counter-staining method is described that gives a contrasting reddish-orange background, curtailing the nonspecific fluorescence in formalin fixed tissues and tissue cultures. Bacteria take up the dye when not of the homologous species. Normal serum conjugated with Lissamine Rhodamine RB200 is added to the fluorescein-labelled antibody, being taken up nonspecifically in the tissue; the specific staining of the fluorescein conjugated antiserum was in no way inhibited or overshadowed in the systems tested. It was generally unnecessary to precipitate or absorb the sera when using this technique. The authors discuss the possible reactions involved and point out that the counterstain apparently plays no part in the serological system to which it has been added.