Specific Immunolabeling Research Articles

Development of gamma-aminobutyric acid immunoreactivity in chick hyperstriatum ventrale and cerebellum: light and electron microscopical observations.

The presence of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the avian hyperstriatum ventrale and dorsal cerebeller vermis was investigated immunocytochemically using a recently characterized antiserum raised against GABA. Tissue from domestic chicks aged from 19 days in ovo to 28 days posthatch was studied with both light and electron microscopy using pre-embedding immunocytochemistry. Basket, stellate and Golgi cells in the cerebellum, considered to be GABA-ergic, exhibited specific GABA-like immunolabelling in perikarya and in axonal and dendritic processes throughout the developmental period investigated. Purkinje cells also exhibited specific GABA-like immunoreactivity in both pre- and posthatch birds but the distribution and intensity of the immunolabelling varied with age and also in its location within the Purkinje neuron. In prehatch birds Purkinje perikarya exhibited heavy immunostaining which was substantially reduced posthatch, whereas the Purkinje primary dendrites remained immunopositive throughout the developmental period. A small population of cells in the medial hyperstriatum ventrale (mHV) were GABA-positive prehatch, but no immunopositive perikarya were evident in any posthatch samples. Small GABA-positive punctate profiles, representing boutons and transversely sectioned axons or dendrites, were present in the neuropil of all age groups studied. Possible reasons for these findings are discussed and it is suggested that the loss of perikaryal immunostaining, both in the cerebellar Purkinje cells and in those of the mHV, may be governed by maturational processes.

Albumin localization in the frog hepatocyte during vitellogenesis as revealed by protein A-gold immunocytochemistry.

The protein A-gold immunocytochemical technique was used to localize albumin in the hepatocyte of the normal male American bullfrog, Rana catesbeiana, and also in the hepatocyte of this animal 8 days after treatment with estradiol-71 beta. Albumin concentration in plasma also was estimated biochemically. In the normal animal, specific immunolabeling for albumin was present in the intracellular compartments involved in protein secretion, i.e., rough endoplasmic reticulum (RER), Golgi apparatus and secretory granules, and also in lysosomes. Density of labeling increased as it progressed along the secretory pathway. In the hepatocyte of the estrogen-treated frog, specific immunolabeling for albumin was also present along the entire secretory pathway and in the lysosomes. Density of labeling over the RER was similar to that seen for this organelle in normal tissue; however, no progressive increase, but rather significant decreases, in labeling density occurred further along the secretory pathway. The biochemical data demonstrated no change in the concentration of plasma albumin in the treated frog, compared with the normal one. These observations localize albumin along its secretory pathway in frog hepatocyte and demonstrate a perturbation in its secretion in response to estrogen treatment.

Enamel protein biosynthesis and secretion in mouse incisor secretory ameloblasts as revealed by high-resolution immunocytochemistry.

Mouse secretory ameloblasts express a number of enamel proteins, which have been divided into amelogenin and enamelin subfamilies. We have used polyclonal antibodies to murine amelogenins to reveal enamel proteins in mouse ameloblasts using the protein A-gold immunocytochemical technique. Specific immunolabeling was detected over the extracellular enamel matrix and over the rough endoplasmic reticulum, the saccules of the Golgi apparatus, and the secretory granules of the ameloblasts. In addition, some lysosome-like granules were also labeled. Only background labeling was obtained over mitochondria, nuclei, cytosol, adjacent odontoblasts, and dentin. Quantitation of the intensity of labeling showed the presence of an increasing gradient along the secretory pathway, which may correspond to the concentration or the maturation of these proteins as they are processed by the cell. These findings indicate that the ameloblast displays an intracellular distribution of its secretory products similar to that of other merocrine secreting cells. The presence of enamel proteins in lysosomes suggests that crinophagy and/or resorption occurs in these cells.

Ultrastructural localization of immunoreactive neurophysins using monoclonal antibodies and protein A-gold.

Using three different monoclonal antibodies against rat neurophysins (5), with protein A-gold as immunocytochemical marker (27), the murid hypothalamoneurohy-pophysial system was studied at the ultrastructural level. Postembedding staining was done on epoxy-embedded sections of supraoptic nuclei and posterior pituitaries. Specific immunolabeling of vasopressinergic and oxytocinergic neurosecretory granules was observed in tissues fixed with glutaraldehyde or glutaraldehyde mixtures (containing paraformaldehyde and picric acid), with or without osmium tetroxide postfixation and with or without sodium metaperiodate oxidation. Some autophagic vacuoles containing lysed neurosecretory granules were also neurophysin immunoreactive. Nonspecific background staining was extremely low. An attempt was made to appraise labeling intensities semiquantitatively by counting gold particles in relation to number of secretory granules per axonal varicosity. Immunoreactivity was measurably influenced by the mode of fixation, sodium metaperiodate oxidation, and titer and affinity of the antibody. The protein A-gold technique using monoclonal antibodies against neurophysins provides a superior means of ultrastructural analysis of the hypothalamoneurohypophysial system, both visually and morphometrically.

In situ identification of immune competent cells in gastrointestinal mucosa: an evaluation by immunoelectronmicroscopy.

The in situ identification of lymphocyte subpopulations by means of immunopathological techniques using specific monoclonal antibodies provides a tool for the study of the gastrointestinal-associated lymphoid tissue (GALT) in health and disease. In this field, monoclonal antibodies have been applied previously using light microscopy and either immunofluorescence or immunoperoxidase; however, these techniques are not sensitive enough to allow precise evaluation of localization of labelling. We describe an immunoelectronmicroscopic method, which defines labelling specificity, since it allows the identification of cells by immunophenotype labelling and ultrastructural markers simultaneously. This in turn allows a better evaluation of the labelled cells and of the relationship between labelled and unlabelled cells. The main features of the method are the use of fresh tissue samples, fixing in paraformaldehyde CaCl2, and the coupling of the immune reaction to an amplification system (avidin-biotin-peroxidase complex). The technique yields a good preservation of cellular ultrastructure, together with a strong and specific immunolabelling. Our results confirm the high specificity of monoclonal antibodies when applied to immunopathology techniques. We confirm the pattern of distribution of various lymphocyte subsets in the jejunal mucosa described by other authors by light microscopy.