Histochemical characterization of the sialic acid residues in mouse colon mucins

The mucins of colonic murine mucus are highly O‐glycosilated sulfosialoglycoproteins. We have characterized the sialylation pattern of oligosaccharide chains of colonic murine mucins by conventional histochemical methods and by lectin histochemistry combined with chemical pretreatments and sialidase digestion. Oligosaccharide chains are strongly sulphated, with an increase of sulfation from the proximal toward the distal colon and a decrease of sialic acid expression and acetylation toward the distal colon. In the goblet cells of proximal colon, sialic acid bound α2,3 to Galβ1,3GalNAc subterminal dimers is diacetylated at C7,C8;C7,C9;C8,C9 or triacetylated at C7,8,9. In the distal colon, sialic acid‐linked α2,3 to Galβ1,3GalNAc subterminal dimers shows reduced O‐acetylation at C7 and/or C8, while acetyl substituents at C9 and at C4 are almost absent. Sialic acid is involved in different essential physiological functions; thus, alterations of its expression and acetylation in oligosaccharide chains of intestinal mucins are generally associated with diseases, such as ulcerative colitis and cancer. Mice may represent a suitable animal model to study alterations of oligosaccharidic chains in colonic mucins and lectin histochemistry combined with chemical pretreatments, and enzyme digestion may be a valuable tool for this study. Our present work may represent a landmark for further lectin histochemical studies to evaluate alterations of mouse colon mucins under different physiological, pathological, or experimental conditions, with possible translational value in humans. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

A histochemical approach to glycan diversity in the urothelium of pig urinary bladder

Intracellular glycans in the urothelium of urinary bladder of 10 adult male Landrace pigs were characterized in situ by immunohistochemical detection of Muc1 mucin by anti MUC1 from rabbit, conventional histochemical techniques (Periodic‐Acid Schiff, Alcian Blue pH 2.5, High‐Iron Diamine), and binding with 13 lectins (PNA, DBA, RCA‐I, WGA, SBA, BSI‐B4, ConA, AAA, UEA‐I, LTA, LFA, MAA‐II, SNA) combined with chemical and enzymatic pre‐treatments (β‐elimination, desulfation and neuraminidase) to gather reference data for this model animal. Muc1 mucin was detected in the secreting granules of superficial cells and the underlying layer of intermediate cells. The secreting granules in both intermediate cells and superficial cells were rich in carbohydrates, with the oligosaccharidic chains mostly O‐linked to proteins. Glycoproteins were prevailing over glycosaminoglycans (GAGs). In both superficial and intermediate cells sulfated and/or sialylated glycans were present, sulfation decreasing in the deeper layers. Lectin‐binding detected presence of terminal sialic acid linked mostly in α2,6 to GalNAc, Gal terminal or subterminal to sulfates, GalNAc, GlcNAc, and Fuc, mostly linked in α1,6, α1,3 α1,4 and α1,2 to GlcNAc or Gal, but not to lactosamine chains. Except for fucosylation, the oligosaccharidic chains in the glycoproteins of the urothelium of pig urinary bladder were similar to those linked to human MUC1, which is fundamental in cell adhesion and immunological processes in the urothelium. The co‐distribution of Muc1 and saccharidic residues suggests that many of them are linked to the glycoprotein.     

Ultrastructural demonstration of exocytosis in the intact rat adrenal medulla

Evidence is presented for morphological proof of exocytosis in the rat adrenal medulla in situ. Techniques were modified to allow perfusion of the intact adrenal gland with secretagogues (or electrical stimulation) followed by tannic acid. Unstimulated specimens demonstrated exocytotic (omega-shaped) profiles filled with flocculent material. This flocculation was also seen in the intercellular space. Stimulation of the adrenal medulla also resulted in the appearance of exocytotic profiles and an accumulation of the flocculent mass. This was often most evident in the subendothelial space. This is the first demonstration of exocytosis in the rat adrenal medulla by electron microscopy. The techniques used in this study will be useful for studying the pathway of secretory products of the adrenal chromaffin cell before they enter the vascular system.     

Involvement of the adrenal glands and testis in gap junction formation via testosterone within the male rat anterior pituitary gland

We investigated the influence of testicular and adrenal androgens on the presence of gap junctions between folliculo‐stellate cells in the anterior pituitary glands of 60‐day‐old Wistar‐Imamichi strain male rats. The animals were separated into six groups: Group A served as the controls and had free access to a normal diet and water, Group B was given a normal diet and 0.9% NaCl for their drinking water as the controls of adrenalectomized groups, Group C was castrated, Group D was adrenalectomized, Group E was both castrated and adrenalectomized, and Group F was also both castrated and adrenalectomized. In addition, the animals of Group F were administered a dose of testosterone that is known to produce high physiological levels of the hormones in plasma. Five rats from each group were sacrificed 1, 2, 3, 4, 5, 6, and 7 days after their respective operation, and the anterior pituitary glands were removed and prepared for observation by transmission electron microscopy. We quantified the number of follicles and gap junctions and calculated the rate of occurrence as the ratio of the number of gap junctions existing between folliculo‐stellate cells per intersected follicle profile. Simultaneous removal of adrenal glands with castration resulted in a significantly decrease in the number of gap junctions, whereas the administration of testosterone to these rats compensated for this change. These observations indicate that the preservation of gap junctions between folliculo‐stellate cells is mainly dependent on androgens from both the testes and adrenal glands in adult male rats. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.     

Histological demonstration of increased vascular permeability in the dura mater of the rat

Neurogenic inflammation of the dura mater encephali has been suggested to play an important role in the pathophysiology of headaches. Although functional studies using extravasation techniques indicate an enhanced permeability of blood vessels after chemical or electrical stimulation of C-fibres supplying the dura mater, histological demonstration of leaky blood vessels is still a problem. We used the vascular labelling method combined with i.v. injection of colloidal silver solution to test the permeability increasing effect of intravenous administration of substance P, topical application of mustard oil or acidic phosphate buffer and local electrical stimulation of the exposed dura mater. Histological characteristics of increased vascular permeability were observed exclusively after mustard oil and acidic phosphate buffer. This observation may indicate different mechanisms of increased vascular permeability involving pinocytosis and formation of interendothelial gaps selectively visualized by the vascular labelling method.     

Technical advances in the sectioning of dental tissue and of on‐section cross‐linked collagen detection in mineralized teeth

Immunohistochemical detection of cross‐linked fibrillar collagens in mineralized tissues is much desired for exploring the mechanisms of biomineralization in health and disease. Mineralized teeth are impossible to section when embedded in conventional media, thus limiting on‐section characterization of matrix proteins by immunohistochemistry. We hypothesized that by using an especially formulated acrylic resin suitable for mineralized dental tissues, not only sectioning of teeth would be possible, but also our recently developed immunofluorescence labeling technique would be amenable to fully calcified tissues for characterization of dentinal fibrillar collagens, which remains elusive. The hypothesis was tested on fixed rodent teeth embedded in Technovit 9100 New®. It was possible to cut thin (1 μm) sections of mineralized teeth, and immunofluorescence characterization of cross‐linked type I fibrillar collagen was selected due to its abundance in dentine. Decalcified samples of teeth embedded in paraffin wax were also used to compare immunolabeling from either method using the same immunoreagents in equivalent concentrations. In the decalcified tissue sections, type I collagen labeling in the dentine along the tubules was “patchy” and the signal in the predentine was very weak. However, enhanced signal in mineralized samples with type I collagen was detected not only in the predentine but also at the limit between intertubular dentine, within the elements of the enamel organ and subgingival stroma. This report offers advances in sectioning mineralized dental tissues and allows the application of immunofluorescence not only for on‐section protein detection but importantly for detecting cross‐linked fibrous collagens in both soft and mineralized tissue sections. Microsc. Res. Tech. 73:741–745, 2010. © 2009 Wiley‐Liss, Inc.     

A study of digital image analysis on the acrylamide derivative monomers induced apoptosis in rat cerebrum

Nowadays, apoptosis is mostly evaluated visually in histological studies. By using the quantitative digital image analysis, this study aimed to investigate the effect of acrylamide‐based monomers (acrylamide [AAm], methacrylamide [MAAm], N‐isopropylacrylamide [NIPAm]) on the cerebrum tissues in rats, which are the most common water‐soluble monomers in the production of polymeric hydrogels used as biomaterials. The Wistar albino rats weighing ~220–240 g were divided into control and three test groups. The control group received 1 mL of saline, and the test groups received 1 mL of aqueous 50 mg/kg/day intramuscular injection of AAm, MAAm, and NIPAm, respectively. At the end of the experiments, brain tissues of all rats euthanized by intramuscular injection of sodium pentobarbital were removed. Terminal deoxynucleotide transferase dUTP nick and labeling (TUNEL) method was applied to brain tissue sections. The monomers have been shown to cause apoptosis due to oxidative stress in cerebrum tissue. Based on apoptosis by tunneling method, quantitative digital image analysis of cell fragments was performed with Olympus cellSens Dimension 1.15 software, and the number, total count area, selected area, average area, and ROI% values of the fragments were found. In addition, the total area and ROI% values of the fragments increased linearly with increasing the molar mass of monomers from the digital image analysis data. Quantitative digital image analysis can facilitate the monitoring of apoptosis caused by the oxidative stress of monomers used in the production of the biomaterials.     

Confocal microscopy with double immunofluorescence staining reveals the functional transient receptor potential vanilloid subtype 1 expressed in myoepithelial cells of human submandibular glands

Myoepithelial cells (MECs) mainly surround acini and intercalated ducts in the human salivary glands. The contraction of MECs provides the expulsive force to promote salivation. We previously found functional transient receptor potential vanilloid subtype 1 (TRPV1) was expressed in rabbit and human submandibular glands and increased saliva secretion. However, it was unknown whether TRPV1 was expressed in MECs of submandibular glands. In this study, we observed the immunoflourescence of TRPV1 was not only located in serous acini and ducts but also surround the basal layer of the acinus and intercalated ducts of human submandibular glands. Double immunofluorescence staining revealed colocalization of TRPV1 with calponin, vimentin, and α-smooth muscle actin, which indicated the myoepithelial expression of TRPV1. Treating submandibular gland tissues with capsaicin, an agonist of TRPV1, substantially increased the phosphorylation of the 20-kDa regulatory light-chain subunit of myosin (MLC(20) ), a crucial molecule for contraction of smooth muscle cells, in MECs. Pretreatment with capsazepine, a specific TRPV1 inhibitor, blocked capsaicin-induced MLC(20) phosphorylation. These results suggest that TRPV1 is expressed in MECs of the human submandibular gland and mediates myoepithelial contraction via a mechanism involving MLC(20) phosphorylation.     

The administration of nonmetabolizable glucose analogues fails to suppress the development of glycogen autophagy in newborn rat hepatocytes

The effects of parenteral administration of glucose, 3‐methylglucose (3MG), or 2‐deoxyglucose (2DG) on the glycogen autophagy were studied in the newborn rat liver using electron microscopy and biochemical methods. The administration of glucose resulted in hyperglycemia and prevented the mobilization of hepatocytic glycogen. It also prevented the development of autophagic vacuoles in general and inhibited the glycogen‐degrading activity of acid α‐1,4‐glucosidase. The nonphosphorylated and not further metabolized glucose analog 3MG also produced hyperglycemia, but increased acid glucosidase. Pretreating the newborns with the β‐adrenergic blocker propranolol inhibited the effects of 3MG. The phosphorylated but not fully metabolized glucose analog 2DG produced similar effects. The administration of xylitol to the newborns already treated with 2DG, suppressed acid glucosidase. The results of this and our previous studies suggest that glucose must be metabolized beyond its phosphorylation step to inhibit acid glucosidase activity. Microsc. Res. Tech. 73:1009–1014, 2010. © 2010 Wiley‐Liss, Inc.     

Immunohistochemistry of neuronal nitric oxide synthase and protein nitration in the striatum of the aged rat

To ascertain the possible implications of the nitric oxide (NO*) producing system in striatal senescence, and by using immunohistochemistry and image-processing approaches, we describe the presence of the enzyme nitric oxide synthase (NOS), the NADPH-diaphorase (NADPH-d) histochemical marker, and nitrotyrosine-derived complexes (N-Tyr) in the striatum of adult and aged rats. The results showed neuronal NOS immunoreactive (nNOS-IR) aspiny medium-sized neurons and nervous fibres in both age groups, with no variation in the percentage of immunoreactive area but a significant decrease in the intensity and in the number of somata with age, which were not related to the observed increase with age of the striatal bundles of the white matter. In addition, NADPH-d activity was detected in neurons with morphology similar to that of the nNOS-IR cells; a decrease in the percentage of area per field and in the number of cells, but an increase in the intensity of staining for the NADPH-d histochemical marker, were detected with age. The number of neuronal NADPH-d somata was higher than for the nNOS-IR ones in both age groups. Moreover, N-Tyr-IR complexes were observed in cells (neurons and glia) and fibres, with a significant increase in the percentage of the area of immunoreaction, related to the increase of white matter, but a decrease in intensity for the aged group. On the other hand, we did not detect the inducible isoform (iNOS) either in adult or in aged rats. Taken together, these results support the contention that NADPH-d staining is not such an unambiguous marker for nNOS, and that increased protein nitration may participate in striatal aging.     

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 Auerbach's 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. © 2009 Wiley‐Liss, Inc.     

Immunohistochemical study on the distribution of sodium-dependent vitamin C transporters in the respiratory system of adult rat

As vitamin C (L-ascorbic acid, VC) is known to be essential for many enzymatic reactions, the study on the transport mechanism of VC through cytoplasmic membrane is crucial to understanding physiological role of VC in cells and the respiratory system. In this regard, the study on the newly identified sodium-dependent VC transporters (SVCTs), SVCT1 and SVCT2, is required in organs that contain high concentration of VC. We have shown the distribution of SVCT proteins in the respiratory system, which has been reported to be one of the organs with a high concentration of VC, using immunohistochemical techniques. In the present study, intense SVCT immunoreactivities (IRs) were mainly localized in the respiratory system epithelial cells. In the trachea, both SVCT1 and 2 were localized in the psuedostratified ciliated columnar epithelium. In the terminal bronchiole, SVCT1 and 2 IRs were mainly observed in the apical portion of the simple columnar epithelium. In addition, SVCT IRs was localized within the cell membrane of some alveolar cells, even though we could not identify the exact cell types. These results provide the first evidence that intense SVCT1 and 2 IRs were found in the apical portion of the respiratory epithelial cells, suggesting that SVCT proteins in the apical portion could transport the reduced form of VC included in the airway surface liquid into the respiratory epithelial cells.     

Role of parietal and principal gastric mucosa cells in the phenomenon of concentration of aluminum and indium

The subcellular behavior of aluminum and indium, used in medical and industrial fields, was studied in the gastric mucosa and the liver after their intragastric administration to rats, using, two of the most sensitive methods of observation and microanalysis, the transmission electron microscopy, and the secondary ion mass spectrometry. The ultrastructural study showed the presence of electron dense deposits, in the lysosomes of parietal and principal gastric mucosa cells but no loaded lysosomes were observed in the different studied hepatic territories. The microanalytical study allowed the identification of the chemical species present in those deposits as aluminum or indium isotopes and the cartography of their distribution. No modification was observed in control rats tissues. In comparison to previous studies describing the mechanism of aluminum concentration in the gastric mucosa and showing that this element was concentrated in the lysosomes of fundic and antral human gastric mucosa, our study provided additional informations about the types of cells involved in the phenomenon of concentration of aluminum and indium, which are the parietal and the principal cells of the gastric mucosa. Our study demonstrated that these cells have the ability to concentrate selectively aluminum and indium in their lysosomes, as a defensive reaction against intoxication by foreign elements.     

Descriptive studies on the tongue of two micro‐mammals inhabiting the Egyptian fauna; the Nile grass rat (Arvicanthis niloticus) and the Egyptian long‐eared hedgehog (Hemiechinus auritus)

The current study aimed to describe the anatomical features of the tongues of two micro‐mammals common in the Egyptian fauna; the Nile grass rat (Arvicathis niloticus), and the Egyptian long‐eared hedgehog (Hemiechinus auritus). The tongues of five adult individuals of each species were excised and processed histologically, histochemically, and morphometrically. Statistical analysis comparing the relative tongue length in both species showed that there was a significant difference, which may correlate with the difference in feeding preferences. Grossly, the Nile grass rat has a dorsal lingual prominence with bifurcated apex while, the long‐eared hedgehog has a median slight elevation with rounded apex. Numerous forms of mechanical and gustatory papillae are scattered along the lingual dorsal epithelium. The histochemical detection of keratin by Holland's trichrome stain showed an intense expression in the case of A. niloticus and mild expression in H. auritus. The framework of the tongue (entoglossum) is supported by either a core of cartilage in H. auritus or bone in A. niloticus which incorporated in the lingual root. The lingual glands also showed marked variation, the Nile grass rat exhibit dense populations of mucous‐secreting glands and lesser populations of serous‐secreting glands, the contrary is true in H. auritus. In conclusion, the micro and macro‐anatomical features of the tongues of both species showed adaptive changes to accommodate the feeding lifestyle. Such type of studies using mammals from different phylogenetic traits and almost have different feeding preferences provide answers to many research questions related to tongue evolution among mammalian vertebrates.     

Age-related changes of NADPH-diaphorase-positive neurons in the rat inferior colliculus and auditory cortex

Nitric oxide (NO) has been implied in age-related changes of the central nervous system (CNS) and the central auditory pathway. The present study was conducted to investigate whether the number of NO-producing cells and their morphometric characteristics in the inferior colliculus (IC) and the auditory cortex (AC) are changed with the increasing age of the subjects. IC and AC sections of adult and senile Wistar rats were studied using the histochemical detection of NADPH-diaphorase activity (NADPH-d), a marker for neurons containing nitric oxide synthase (NOS). Our results showed a decreased area of the somas of NADPH-d-positive neurons in the dorsal cortex (DC) of the IC and a diffuse loss of NADPH-d-positive neurons in the senile IC and primary cortical auditory area (Te1). However, an increased number of NO-producing cells have been shown by other authors in different parts of the ageing auditory pathway and CNS. It seems that age-related changes in NADPH-d-positive cells may follow a region-specific route. These changes may be related to hearing impairments with increasing age.     

Involution dependent changes in distribution and localization of bax,survivin, caspase‐3, and calpain‐1 in the rat endometrium

The endometrial layer of the uterus is characterized by continuous cycle of cell growth and apoptosis in response to hormonal changes. Apoptosis is regulated by several apoptotic regulators, but their significance in involuting uterus has not been well understood. For that reason, aim of this study was to investigate possible role of apoptosis‐related proteins (bax and survivin) and enzymes (caspase‐3 and calpain‐1) in the involuting uterus of the rat, using immunohistochemistry. Our results indicated cytoplasmic and nuclear immunostaining for bax, caspase‐3, calpain‐1 and survivin proteins were found in the endometrial epithelium and stromal cells such as fibroblasts, mast cells and macrophages, and blood vessels; however, calpain‐1 immunoreactivity in the endometrial fibroblast was quite weak or absent. Supranuclear punctate bax immunolabelling was also observed in the endometrial fibroblasts and luminal and glandular epithelial cells from days 1st and 3rd following parturition, respectively. Although survivin was localized in the apical cytoplasm underneath the apical membrane of the luminal epithelium on the 1st and 3rd days, it was also localized in the apicolateral membrane and basal cytoplasm on the 10th and 15th days of involution. Immunostainigs demonstrated that expression patterns of all examined proteins varied with structural changes in the luminal epithelium, and number of immunopositive fibroblasts for bax, caspase‐3 and survivin increased with advance of postpartum days and reached a maximum on postpartum days 10 and 15. These results suggest that the process of postpartum involution of endometrium may be regulated by apoptotic and non‐apoptotic activity of bax, caspase‐3, calpain‐1, and survivin. Microsc. Res. Tech. 79:285–297, 2016. © 2016 Wiley Periodicals, Inc.     

Autophagosomal glycogen‐degrading activity and its relationship to the general autophagic activity in newborn rat hepatocytes: The effects of parenteral glucose administration

The effects of the administration of parenteral glucose on the postnatal glycogen autophagic activity and its relationship to the general autophagic activity, were studied in newborn rat liver using electron microscopy and biochemical methods. Glucose abolished the normal postnatal hypoglycemia and preserved the hepatocytic hyaloplasmic glycogen to the levels of birth. It also inhibited the normal postnatal increase in the number and volume of autophagic vacuoles. Glucose especially decreased the rate of postnatal development of the glycogen‐containing autophagic vacuoles. This decrease was greater than that of the autophagic vacuoles in general. In the control animals at the age of 6 h, the total volume of the glycogen‐containing autophagic vacuoles accounted for 87% of the autophagic vacuoles in general, whereas in the glucose‐treated animals of the same age, for only 62%. The results of this and previous studies support the view that the general autophagic activity that develops in the immediate postnatal period in rat hepatocytes is mainly expressed as glycogen autophagic activity selectively inhibited by glucose. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

The revolution of counting "tops": two decades of the disector principle in morphological research

Twenty years have passed since the publication of the seminal paper enunciating the disector principle by an author using the pseudonym D.C. Sterio. During this time, methods based on the revolutionary principle of counting "tops" have become progressively better known and have been included in several commercially available systems for quantitative morphology. Analysis of the number of published studies citing Sterio's paper on the ISI Web of Knowledge database showed that its scientific "impact factor" has almost continuously risen since its publication, indicating the growing knowledge about disector-based methods in the various scientific fields where morphological quantification is required. This report briefly reviews the first two decades of disector use, pointing to its advantages as well as to shortcomings that have recently been addressed in critical papers and have given rise to a lively debate on the role of counting tops in quantitative morphology today.     

Cell-surface ultrastructural changes during the in vitro neuron-like differentiation of rat bone marrow-derived mesenchymal stem cells

The neuron-like differentiation of bone marrow-derived mesenchymal stem cells (BMMSCs) has been extensively studied. However, the alternations of the cell-surface ultrastructures and the membrane tension/reservoir of the cells during this differentiation process are poorly understood. Therefore, atomic force microscopy (AFM) was utilized in this study to observe the cell-surface ultrastructural changes among rat bone marrow-derived mesenchymal stem cells (rBMMSCs), partially differentiated cells, and fully differentiated neuron-like cells. By analyzing the stiffness of plasma membranes, lamellipodial extensions, average heights of small membrane protrusions and relatively larger uplifted structures, and peak-peak spacing among protrusions and/or uplifted structures, we found that the membrane reservoir may potentially decrease upon the differentiation from rBMMSCs to partially differentiated cells and to fully differentiated neuron-like cells. The results may help to better understanding the membrane tension of various types of cells and related biological processes, such as membrane traffic, cell adhesion, motility, differentiation, among others. The data also implies that AFM may be a useful tool for evaluating membrane reservoir by imaging cell-surface ultrastructures.     

Three-dimensional visualization and physiologic evaluation of bile canaliculi in the rat liver slice by confocal laser scanning microscopy.

We evaluated the morphology and physiologic function of the bile canaliculi (BC) in the rat liver slice (RLS) by confocal laser scanning microscopy (CLSM). Lucifer yellow (LY) dye was injected into the RLS, and the distribution of LY was serially evaluated. After the injection of LY, hepatocytes were initially visualized, followed by visualization of the BC. There was no significant difference in the distribution of LY between zones 1 and 3 in the hepatic lobule. In zone 1, the reticular distribution of the BC was observed, whereas the part of BC was linearly visualized in zone 3 along the course of sinusoids. When changes in the bile canalicular fluorescence (BCF) were serially evaluated, the BCF was decreased to the minimal level (88% of the value obtained immediately after the LY injection) 10 min after the LY injection, and it tended to increase thereafter. The intralobular hepatocyte fluorescence (ILHF) was decreased to 58.9% of the initial value during the first 40 min. However, the ILHF was transiently increased 30 min after the LY injection, suggesting the possibility of reabsorption of LY by hepatocytes. Three-dimensional (3-D) reconstruction images of the BC facilitated the evaluation of the stereoscopic structure of BC. Confocal laser scanning microscopy facilitated the evaluation of structures and physiologic function of the BC.