Serial semithin sections in immunohistochemistry: techniques and applications.

Immunostaining of semithin sections is a valuable tool in biomedical research; however, this method is rather rarely used by histologists. To overcome the apparent reservations concerning this method, the present report describes a technique which is rather simple, largely standardized, and very useful in investigative endocrinology. The technique includes the following steps: snap-freezing and freeze-drying of tissue specimens, embedding in Araldite, preparation of serial semithin sections, removal of the resin from tissue sections, immunostaining with Sternberger's peroxidase antiperoxidase (PAP) technique, and analyses of the sections by various microscopical techniques which in part optically enhance immunoreactive sites. In addition to a detailed description of the method, examples for its applications are given, including concomitant investigations of the same cells by empirical staining, immunostaining, and fluorescence histochemistry of biogenic monoamines; colocalization of multiple peptides to the same cells and corresponding specificity controls; three-dimensional reconstructions based upon immunostained serial semithin sections; quantitative (computer-assisted) determinations of immunoreactivities. Because of the advantages offered by immunostained serial semithin sections as well as the vast field of applications, the method described is recommended for routine use. Concomitantly this method covers the gap between conventional light microscopy (paraffin sections) and electron microscopy.