We investigated non-specific staining in a catalyzed reporter deposition (CARD) reaction and improved its blocking methods in supersensitive immunohistochemistry, based on our simplified catalyzed signal amplification (CSA) system (Hasui et al. 2002). In the CARD reaction using biotinyl tyramide, non-specific staining could be reduced by pretreatment with a casein solution or 3% bovine serum albumin (BSA)-phosphate buffer saline (PBS) with 0.1% Tween 20. In the CARD reaction using FITC-labeled tyramide, non-specific staining could be blocked by pretreatment with 0.3% BSA-PBS with 0.1% Tween 20 or 3% polyethylene glycol-PBS with 01% Tween 20. Thus, our new simplified CSA system features: 1) destruction of the endogenous peroxidase activity; 2) blocking of the nonspecific reaction of the primary antibody; 3) a primary antibody reaction; 4) blocking of the non-specific reaction of the polymer reagent by casein treatment; 5) a polymer reaction; 6) blocking of the non-specific reaction of CARD reaction by casein treatment; 7) a CARD reaction; and 8) detection of deposited tyramide. This new system proved useful for detecting an extremely low amount of antigen in the endogenous biotin-rich tissues such as the gastrointestinal tract and liver. By this method, the Ki67 antigen in the G1 phase cell cycle could be detected and a metabolic disorder of the Ki67 antigen was implicated in a carcinoid tumor in the stomach. We believe that this new simplified CSA system represents a new standard of supersensitive immunohistochemistry for use in light-microscopic investigation.

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Serotonin (5-HT) has been a candidate for neurotransmitters in cutaneous type I mechanoreceptors (i.e., Merkel cell-nerve endings). Although recent electrophysiological studies have suggested the presence of the 5-HT2 and 3 receptors in the Merkel cell-nerve endings, the histological localization of these receptors are obscure. We thus immunohistochemically examined the presence of 5-HT1, 2, 3 receptors in Merkel cell-nerve endings in sinus hair follicles of the rat whisker pad. We also studied the immunohistochemical localization of the 5-HT transporter to confirm the site of 5-HT secretion. For this purpose, we used antibodies for the 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2C and 5-HT3 receptors, and for the 5-HT transporter, as well as antibodies for cytokeratin 20 (as a marker of Merkel cells) and neurofilament H (a marker of type I sensory nerve terminals). The immuno-stained sections were analyzed under a laser-scanning microscope. It was found that the sensory nerve terminals in the Merkel cell-nerve endings showed strong positive immunoreactions of 5-HT1A and 1B receptors but not 5-HT2A, 2C, and 3 receptors. Furthermore, both the Merkel cells and related axon terminals showed strong immunoreactions of the 5-HT transporter. These findings support the idea that 5-HT molecules are released from the Merkel cells during mechanical reception and indirectly regulate neural actions of sensory neurons via 5-HT1 receptors. The localization of the 5-HT transporter found in this study also suggests a possibility that axon terminals in the Merkel cell-nerve endings also release 5-HT.

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To clarify the possible role of nitric oxide (NO) induced in primary sensory neurons after peripheral axotomy, NO synthase (NOS) immunohistochemistry was carried out on rat L5 dorsal root ganglia after sciatic nerve ligation. The results were compared with the expression of 27-kDa heat shock protein (HSP27), a neuroprotective molecule. In intact animals, NOS-immunoreactive neurons represented about 2% of all dorsal root ganglion (DRG) neurons, whereas HSP27-immunoreactive neurons comprised about 14%. After sciatic nerve ligation, both neurons increased, in number and immunoreactivity, reaching a maximum at 2weeks, when NOS- and HSP27-immunoreactive neurons represented about 33 and 66%, respectively. NOS-immunoreactive neurons then remained unchanged until 7 weeks although HSP27-immunoreactive neurons showed a slight decline. The increased NOS-immunoreactive neurons were preferentially small (100—500μm²) and coexpressed with HSP27 (about 87%). On the other hand, in the proximal stump of sciatic nerves, numerous NOS-immunoreactive fibers with a regenerative profile appeared transiently (2—4weeks). At higher magnification, an axonal sprout from the NOS-immunoreactive small DRG neurons was found to form a basket-like structure (or basket) mostly around the cell body of NOS-negative large neurons. Retrograde labeling with a fluorescent tracer showed that both neurons sent peripheral axon collaterals to the sciatic nerve. The appearance of this unique structure was most prominent after depletion of the NOS-immunoreactive regenerating fibers in the sciatic nerve (at 7—9weeks). The findings suggest that NO might be involved in not only axonal regeneration but also the rewiring of two classes of DRG neurons after peripheral nerve injury.

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Recent studies have suggested that an ischemia/reperfusion (I/R) injury enhances the expression of costimulatory adhesion molecules on the vascular endothelium. In the present study, we investigated the protective effects of resveratrol, a phenolic product, on the renal function and expression of CD86 in rat kidneys with I/R injury. Wistar rats were divided into four groups;1) an I/R group with right nephrectomy and 1-hour clamping of the left renal pedicle; 2) a vehicle group, I/R plus 10% ethanol (0.1ml/kg/day) administered by intra-peritoneal injection from day —1 through to 7; 3) a resveratrol group, I/R plus 4mg/kg/day of resveratrol; and 4) a sham group. Blood samples were obtained via the tail vein at 1 day before, and 1, 3, and 7 days after the operation (day 0) for the measurement of serum creatinine (Scr) levels. The expression of CD86 protein was analyzed by immunofluorescence staining, and the level of CD86 messenger RNA (mRNA) was evaluated quantitatively by a real-time reverse transcription-polymerase chain reaction (RT-PCR) in the renal cortex at day 3. Scr levels of the resveratrol group were significantly lower than those of the I/R and vehicle groups on days 1 and 3 after the operation. From the immunohistochemical study, the expression of CD86 in the glomerular endothelium and peritubular vessels was found to be attenuated in the resveratrol group compared with the I/R or vehicle group. In the resveratrol group, the CD86 mRNA level was significantly lower than that in the I/R or vehicle group, and it was significantly decreased by about one fifth of that in the sham group. Our results suggest that resveratrol markedly reduces renal dysfunction and attenuates the mRNA and protein expression of CD86 following I/R injury.

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The three-dimensional ultrastructure of the filamentous glycocalyx of the brush border in the mouse small intestine was successfully demonstrated by high resolution scanning electron microscopy (SEM). The specimens were fixed with 2% glutaraldehyde in a 0.1M phosphate buffer (pH7.4), and rinsed with buffered solutions with differently adjusted pH values (pH3.0, 7.0 or 11.0). They were then osmicated, dried, spatter-coated with gold (1.0-1.5nm), and observed under a high resolution SEM. The glycocalyx on the luminal surface of the intestinal villi covered the top of the microvilli of the epithelial cells and were well preserved in the specimens treated with an alkaline buffer (pH11.0). The glycocalyx was observed as filamentous structures, 7 to 15nm thick in diameter. These filaments repeatedly branched and anastomosed with neighboring ones to form an actual network or plexus as a whole, in contrast with superimposed images in transmission electron microscopy (TEM) which suggested that such anastomoses were pseudo-networks. The filaments thickened globularly at the sites of the filament bifurcation or branching. On the other hand, specimens rinsed with an acid or neutral buffer showed no glycocalyx on their microvilli, whose naked top had knob-like structures. Thus, the pH values of the washing buffer solutions were considered to affect the preservation of the surface coat due to molecular characteristics.

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The localization of calbindin-D28K (CB) was studied immunocytochemically in laminae I and II of the dorsal horn and in spinal ganglia in the chicken, and compared with the distribution of substance P (SP) using double immunolabeling. At the light microscopic level, CB immunoreactivity was observed most intensely in the lamina II using the avidin-biotinylated peroxidase complex (ABC) and immunofluorescence methods. At the electron microscopic level using the ABC method, CB immunoreactivity was observed in the following three neuronal elements: 1) the scalloped central terminal with many dense-cored vesicles (DCVs) in the synaptic glomerulus;2) some vesicle-containing dendrites (VCDs) inside or outside the synaptic glomerulus; and 3) some axon terminals outside the synaptic glomerulus. The CB-immunoreactive (IR) VCDs in the synaptic glomerulus often formed reciprocal synapses with the central terminal. Strong immunoreactivity was observed at the postsynaptic membrane of CB-IR elements. Double immunofluorescence and immunolabeling methods at the electron microscopic level showed that CB and SP colocalized in the scalloped central terminal with DCVs of the synaptic glomerulus. Almost all SP-IR neurons in the spinal ganglion revealed the coexistence of CB in serial sections in the chicken. In light of previous biochemical and physiological reports, our findings suggest that CB - coexisting with SP - plays an important role in the control of pain transmission through its strong Ca²⁺-buffering action in the chicken.

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Cold ischemia-warm reperfusion injury of liver grafts has been investigated thoroughly, but its underlying mechanism remains poorly understood. Here we show that autophagy is involved not only during cold preservation but also during warm reperfusion following transplantation. Immunohistochemistry using an antibody against LC3, a microtubule associated protein 1 light chain 3 and a marker of autophagosomes, showed dot-like weak staining in hepatocytes of rat liver grafts during cold preservation. Since University of Wisconsin solution for graft preservation lacks amino acids, the induction of autophagy in hepatocytes was similar to that under starvation conditions. Intense immunopositive punctate structures were detected abundantly in the hepatocytes 30min after the beginning of reperfusion. LC3-positive granules were often co-localized in ED2-positive Kupffer cells at 60min of the reperfusion phase. The molecular form of LC3 was mainly LC3-II, a membrane-bound form, during reperfusion, especially at 30min of the phase. Electron microscopic examination demonstrated numerous vacuolar structures in hepatocytes at 30min of the reperfusion period, while some hepatocytes with such vacuolar structures were present in the sinusoidal lumen. At the late stage of the reperfusion period, Kupffer cells contained phagocytosed cells that possessed numerous autophagic vacuoles/autolysosomes and nuclei with condensed chromatin. Our results showing the presence of autophagic vacuoles/autolysosomes in hepatocytes of liver grafts after the start of reperfusion suggest that warm reperfusion acted as a stress stimulus to hepatocytes. Moreover, the stress response of hepatocytes may be involved in their degeneration process.

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The ultrastructure of C-banded human metaphase chromosomes was studied by the combined use of light microscopy and atomic force microscopy (AFM). Light microscopy of the C-banded chromosomes showed that the centromeric regions of all chromosomes except the Y chromosome were positively stained. AFM further revealed that the C-positive region was higher than the C-negative region. The area of the C-positive region was specific depending on each chromosome; it ranged from the centromere to the proximal end of the long arm in chromosome 1, while it was restricted to the centromere in chromosomes 2 and 3. At higher magnification, chromatin fibers about 50nm thick were clearly shown in the entire length of the chromosomes. In the C-positive region, these chromatin fibers were densely packed, while chromatin fibers were loosely packed with gentle twisting in the C-negative region. These AFM findings suggest that certain factors related to the chromatin fiber compaction remain in the C-positive region even after successive C-banging treatment.

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