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Introduction of Tyramide Signal Amplification (TSA) to Pre-embedding Nanogold-Silver Staining at the Electron Microscopic Level

The tyramide signal amplification (TSA) technique has been shown to detect scarce tissue antigens in light and electron microscopy. In this study we applied the TSA technique at the electron microscopic level to pre-embedding immunocytochemistry. This protocol was compared to the non-amplified protocol. With the TSA protocol, the labeling of GM130, a cis-Golgi matrix protein, was tested in a cell line and found to be highly sensitive and more enhanced than that with the simple protocol. Moreover, the gold particles were well localized to the cis-side of the Golgi apparatus in both the TSA and the simple protocol.

Distribution and three-dimensional appearance of the interstitial cells of Cajal in the rat stomach and duodenum

The relationship between the interstitial cells of Cajal (ICC) and enteric nerves or smooth muscles cells is not fully defined. Presently, distribution and appearance of ICC in the rat stomach and duodenum was studied by immunohistochemistry, electron microscopy, and three‐dimensional reconstruction. c‐kit expressing ICC were regularly observed in the Auerbachs myenteric plexus (AP) of the stomach and duodenum. ICC in stomach and duodenum muscle layers was dissimilarly distributed. c‐kit immunoreactive cells were sparsely distributed in the stomach circular muscle layer but were abundant in the duodenum deep muscular plexus (DMP). Electron microscopy revealed that stomach ICC‐AP were irregular ovals with few cytoplasmic processes, and possessed an electron‐dense cytoplasm, numerous mitochondria, intermediate filaments, and caveolae. Duodenum and stomach ICC‐AP were similar in appearance. Ultrastructure observations and three‐dimensional reconstructions revealed ICC‐AP processes wrapping the nerve fibers and projecting into the space between smooth muscle cells. While ICC‐AP was occasionally close to enteric nerves or smooth muscle cells, no connections were observed. ICC‐DMP in duodenum was elongated and adopted the same cell axis orientation as the circular muscle cells. Unlike ICC‐AP, ICC‐DMP formed gap junctions with smooth muscle cells and had close contact with nerves. These results indicate that ICC‐AP is regularly distributed in stomach and duodenum, while ICC‐DMP is exclusively located in the duodenum. ICC‐DMP, which possess gap junctions and closely contacts nerves, may participate in neuromuscular transmission. Microsc. Res. Tech. 2009.

Postnatal expression and distribution of Refsum disease gene associated protein in the rat retina and visual cortex: effect of binocular visual deprivation.

Previously, phytanoyl‐CoA alpha‐hydroxylase‐associated protein 1 (PAHX‐AP1) was isolated as a novel neuron‐specific protein to interact with Refsum disease (RfD) gene PAHX. Its expression in the brain increased after eyelid opening, and the elevated level was maintained through adulthood. In this report, to verify the hypothesis that light could trigger this increase, we have examined the developmental distribution pattern of PAHX‐AP1 in rat retina and visual cortex, and changes of its expression by binocular deprivation. Northern blot analyses demonstrated PAHX‐AP1 expression reached its highest level in the visual cortex and eyeball at 4 weeks after birth, and these levels were maintained through adult life. Two weeks after visual deprivation, its expression in the eyeball and visual cortex decreased compared with the control. In situ hybridization analyses of the retina showed that PAHX‐AP1 expression was limited to the ganglionic cell layer at 10 days after birth, but expressed in the inner nuclear cell layer and extended to the outer nuclear cell layer at 2 and 3 weeks after birth, respectively. Two weeks after visual deprivation, however, it decreased in the ganglionic and inner nuclear cell layer, and disappeared in the rod and cone cell layers. In the visual cortex, strong signals of PAHX‐AP1 were detected in layers IV and VI, and II–VI at 10 days and 2 weeks after birth, respectively. Its expression decreased after 2 weeks of visual deprivation. These results indicate that visual stimulation is essential for the maintenance of PAHX‐AP1 expressions in the retina, especially in the rod and cone cell layers, and visual cortex, and suggest that PAHX‐AP1 may be involved in the developmental regulation of the photoreceptors function.