Use of an improved liquid drop retainer for combining immunofluorescence and peroxidase-antiperoxidase techniques

An improved "liquid drop retainer" was made by gluing a circular cover glass within a selected ceramic ring of a serological ring slide. One to three small sections of tissue mounted within corresponding circles on an ordinary microscopic slide could be immunostained with primary antisera when placed upside-down on the liquid drop retainer. After rinsing in the buffer, the sections were placed upside-down over the liquid drop retainer containing fluorescein isothiocyanate-conjugated immunoglobulin and photomicrographs were taken by placing the inverted sections over the liquid drop retainer. After taking the photomicrographs, the sections were incubated in peroxidase-antiperoxidase complex and placed upside-down over the liquid drop retainer. These liquid drop retainers provided the following advantages in immunocytochemical staining: (1) greatly reduced volumes of antibody solutions to micro-amounts (0.08-0.12 ml); (2) allowed simultaneous localization of different antibodies in adjacent serial sections on the same slide; and (3) allowed immunofluorescence and peroxidase-antiperoxidase techniques to be done on the same section without any damage by immunofluorescence photography before using the peroxidase-antiperoxidase technique.