Ultrathin frozen sections can be cut smoothly from many fixed and appropriately treated specimens. To use such sections for immunochemical localization of intracellular antigens, fixa ion conditions must be selected to optimize at least three variables, namely, preservation of ultrastructure, preservation of antigenicity and retention of accessibility of the antigen to the antibody. Furthermore, staining of the sections must be such that both the immunolabels and structures are clearly recognized. Our efforts to attain these goals are described in relation to their historical background. Although there are still problems to be solved and improvements to be made, we now consider that cryoultramicrotomy has reached the stage of being useful in studying many questions which will not be easily approached otherwise.

Immunochemistry on ultrathin frozen sections

Adenohypophysis: ultrastructural cytochemistry. A review.

The enzyme tyrosine hydroxylase (TH) was immunohistochemically localized by the peroxidase-antiperoxidase method in rat to chromaffin cells of the adrenal medulla, large neurons and small darkly staining cells of the superior cervical ganglia and noradrenergic and dopaminergic neurons in brain. As compared with the conjugated peroxidase or immunofluorescence techniques, the peroxidase-antiperoxidase method gave the most selective and specific cytoplasmic localization of TH antisera in every tissue examined. The peroxidase staining with the TH antisera was more intense in dopaminergic than in noradrenergic neurons of the central nervous system. While TH was visualized in cell bodies of both dopaminergic and noradrenergic neurons, it could only be detected in axons and terminals in the dopaminergic system. The perikarya of noradrenergic neurons could be distinguished from dopaminergic neurons by the immunohistochemical demonstration of the enzyme dopamine-beta-hydroxylase only in the former.

Cellular localization of tyrosine hydroxylase by immunohistochemistry.

Almost a decade has passed since the initial application of enzymes as labels in immunohistocytochemistry. During these years, the peroxidase-labeled antibody method has been used successfully by several laboratories for localizing antigens at both the light and electron microscopic levels. Recent improvements in the methodologies associated with the conjugation of peroxidase to immunoglobulin, the fixation of antigens, and the penetration of peroxidaselabeled antibodies into tissues should greatly facilitate successful application of the method.

Recent progress in the peroxidase-labeled antibody method.

The technique involving use of unlabeled antibody and the peroxidase-antiperoxidase complex was used to identify the adrenocorticotropin (ACTH)-secreting cell in the anterior pituitary lobe of the rat and to localize ACTH in it electron microscopically in ultrathin sections The ACTH cell is star-shaped, with processes extending around other cells, and contains secretory granules of a maximal diameter of 300 mµ arranged peripherally along the plasma membrane Stain was observed on secretory granules, around them, in the Golgi complex and in rough endoplasmic reticulum One day after adrenalectomy, the ACTH cell is degranulated and the staining intensity of its remaining granules and cytoplasm is decreased, suggesting release of ACTH stores If cortisol is given 6 hr after adrenalectomy, 18 hr later the ACTH cells are well granulated and the granules stain more intensely than normal In addition, staining around the granules and throughout the cytoplasm is more intense, suggesting that an early effect of c

Electron microscopic study of the adrenocorticotropin-producing cell with the use of unlabeled antibody and the soluble peroxidase-antiperoxidase complex.

Peroxidase-labeled antibody method was used to localize tissue antigens directly on ultrathin sections. For the demonstration of the method, luteinizing hormone, growth hormone and prolactin were localized on ultrathin sections of methacrylate-embedded anterior pituitary gland of the rat. The hormones were localized in secretion granules and in endoplasmic reticulum, but not in nucleus or mitochondria. This approach totally eliminates the problem of penetration of antisera through tissues and permits better immunologically controlled experiments by using serially sectioned materials.

Yasuyuki Kawarai

Papers

Localization of tissue antigens on the ultrathin sections with peroxidase-labeled antibody method.