Although hydrophilic acrylic resins including LR White have been widely utilized as embedding media for immunocytochemical use, the constituents of tissues are often extracted by the resin monomer during the infiltration process of the embedment, resulting in a discernible impairment of the ultrastructure when the tissue is weakly fixed only with aldehydes. To minimize the extraction by the resin monomer, the embedding procedure with LR White resin was reexamined in the present study. Among the treatments tested, a partial dehydration with 70% ethanol containing 2% phosphotungstic acid (PTA) well preserved the ultrastructure of the pituitary tissue without spoiling the antigenicity of LHβ and other representative markers for the Golgi apparatus. In addition, treatment with 1% tannic acid (TA) prior to the dehydration described above synergistically improved both the ultrastructure and antigenicity of the tissue so that the orientation of the Golgi apparatus could be determined by double immunogold labeling with commercially available anti-GM130 and anti-TGN38 antibodies. The ultrathin sections from the LR White-embedded tissue treated with TA and dehydrated in 70% ethanol containing 2% PTA also enhanced contrast without conventional heavy-metal staining with uranyl acetate and lead citrate. Our findings further suggest that the precipitation of TA and PTA protected the tissue from being extracted during the embedment, probably because an insoluble complex was transiently formed with the constituents of the tissue. This simple modification of the LR White embedment can extend the application of post-embedding immunocytochemistry as an alternative to pre-embedding immunolabeling with frozen ultrathin sections.

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Tight junctions regulate paracellular permeability, create the luminal fluid microenvironment of blood vessels and the digestive tract, and also form the protective barrier in the stratified epithelium including the epidermis. Claudins are the integral membrane proteins at tight junctions and form a multigene family composed of at least 24 members, but knowledge of the subcellular localization of each claudin is still fragmentary. We performed RT-PCR for fifteen claudin species to examine the mRNA expression in various mouse tissues, and focused on investigating the subcellular localization of claudin-10 and -15 by immunofluorescence microscopy in various rat tissues. Neither claudin-10 nor -15 was detected in vascular endothelial cells in most tissues, and these claudins were restricted to the vasa recta in the kidney medulla. Both claudins were also detected at apical tight junctions in the epithelium of the jejunum with no intensity gradients along the crypt-to-villus axis. However, both claudins were expressed only in the basal half of the crypt epithelium in the colon, showing obvious gradients along crypt-to-surface axis. Moreover, claudin-10 showed the ectopic subcellular localization where tight junction strands do not exist. Claudin-10 was detected along the entire lateral membranes of acinar cells in addition to the apical tight junctions in exocrine glands, and in the cytoplasm of basal cells in the stratified epithelium including the dorsal skin and cutaneous stomach. These heterogeneous distributions of claudin-10 and -15 in tissues may be related to the differences in paracellular permeability among tissues.

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Radial glial cells are transiently bipolar cells in the developing central nervous system, best known for their role in guiding migrating neurons. The aim of the present study was to investigate phenotypic characteristics of these bipolar precursor cells in a mixed glial cell culture system derived from the rat neonatal spinal cord. Morphological characterization was assessed by cell-specific immunocytochemical markers (nestin, vimentin, 3CB2) and transmission electron microscopy. Our study yielded substantial evidence showing that the bipolar cells exhibit immunocytochemical and ultrastructural features of radial glial cells. Immunohistochemistry of the neonatal rat spinal cord using the same cell-specific markers suggested these cells are likely derived from the subependymal zone, ventral commissure, and dorsomedial septum. We believe our data recommend this mixed glial culture system to be a valuable tool in studying radial glial cells in vitro.

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Mice homozygous for klotho gene deletion are well established aging models as they mimic certain aspects of human senescence e.g. osteoporosis. Induced senescence may affect cellular functions and alter the histological properties of the extracellular matrices. The present study examined the histological and ultrastructural features of osteocytes and the surrounding bone matrix in klotho-deficient mice. As expected, osteoblasts showed a flattened shape with a weak immunoreactivity for alkaline phosphatase, and the bone matrix contained many empty osteocytic lacunae. The walls of both normal and empty lacunae were intensely immunopositive for osteopontin and dentin matrix protein-1, but featured an inconsistent immunoreactivity for osteocalcin and type I collagen. Not surprisingly, TUNEL-positivity, indicative of apoptosis, was found in many osteoblasts, osteocytes, and bone marrow cells of the klotho-deficient mice. In transmission electron microscopy, an amorphous matrix containing non-collagenous organic materials was recognizable around osteoblasts and in the osteocytic lacunae. Some osteoblasts on the bone surface featured these amorphous materials in vacuoles associated with their trans-Golgi network, indicating that, under klotho-deficient conditions, they synthesize and secrete the non-collagenous structures. Some osteocytes displayed pyknosis or degenerative traits. Thus, our findings provide histological evidence that klotho gene deletion influences the spatial distribution of osteocytes and the synthesis of bone matrix proteins in addition to the accelerated aging of bone cells.

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The role of Merkel cells in type I cutaneous mechanoreceptors remains enigmatic though mechanical transduction or neuromodulation function has been proposed. It has been shown that mammalian Merkel cells express immunohistochemical reactions of met-enkephalin, VIP, substance P, and CGRP, though the reactivity differs between species. If any one of these peptides acts as a transmitter or modulator for Merkel nerve terminals, these structures must have a specific receptor for the substance. We therefore studied the immunohistochemical localization of the above-mentioned neuropeptides and their receptors in Merkel cell-nerve endings in rat whisker pads. Specimens were doubly stained with polyclonal antibodies to neuropeptides and their receptors combined with a monoclonal antibody to cytokeratin 20, which was used for the labeling of Merkel cells. Merkel cells in the rat sinus hair follicles showed positive immunoreactions for all peptides studied, whereas the immunoreactions of receptors to these peptides were localized on Merkel cell membranes but not on the axon terminals. These results suggest that neuropeptides released from Merkel cells act on Merkel cells themselves by an autocrine mechanism.

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Neurotrophin-3 (NT3) and neurotrophin-4 (NT4) affect the survival and maintenance of central and peripheral neurons. Using an immunohistochemical method, we examined whether the taste bud cells in the circumvallate papillae of normal mice expressed NT3, NT4, and their respective receptors TrkC and TrkB, and if so, what type of cells in the taste buds expressed them. Double immunostaining for either of them and PGP 9.5, NCAM, or gustducin was used to determine which cell types expressed which neurotrophins and receptors. Normal taste bud cells expressed NT3, NT4, and the TrkB receptor, but not TrkC. The percentage of NT3-immunoreactive cells among all taste bud cells was 89.0%, that of NT4-immunoreactive cells, 58.6%, and that of TrkB-immunoreactive cells, 80.8%. Almost none of the NT4-immunoreactive cells were reactive with anti-PGP 9.5 or the anti-NCAM antibody, but they could be stained with anti-gustducin, revealing that NT4-immunoreactive cells were contained only in the type-II - and possibly type-I - cell population. On the other hand, NT3-, and TrkB-immunoreactive cells included type-III cells, together with type-II, -I, and basal cells, because they were positive for PGP 9.5 and gustducin. We conclude that NT4 may exert trophic actions on all types of taste bud cells by binding to their TrkB receptors, and NT3 may also have a similar, though negligible role.

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