Lowicryl K4M Research Articles

Pre- and Post-embedding Immunogold Labeling of Tissue Sections.

Despite the improved resolution capacities of fluorescence microscopy over the last 20 years, localization of specific proteins at the ultrastructural level with gold-conjugated antibodies remains a valuable technique in the cell biological tool chest. Ultrastructural immunolocalization of specific proteins in tissues rather than in cultured cells is often advantageous because, in tissues, the interactions between different cell types and with the extracellular matrix are maintained. Here, we describe two immunogold labeling procedures to localize at the ultrastructural level one or more proteins. In the first procedure (pre-embedding), micrometer-thick tissue cryostat sections are immunostained prior to embedding for obtaining ultrathin sections suitable for TEM, while in the second procedure (post-embedding), tissues are embedded in a hydrophobic resin such as Lowicryl K4M and ultrathin sections are first obtained and then immunolabeled. While the former method is better at generating strong immunolabeling, the latter is better at preserving ultrastructure.

Presence of F-actin in sperm head of Armadillidium peraccae (Isopoda, Oniscidea)

Sperm of Armadillidium peraccae have been examined with cytochemical and immunocytochemical methods for fluorescence and electron microscopic visualization of cytoskeleton components. Sperm incubation in an antibody anti-β-tubulin shows only the presence of two centrioles located in the cytoplasmic region above the nucleus; no other microtubules are present in the sperm head. Instead, fluorescence microscopy of sperm incubated in FITC-phalloidin allowed to detect the presence of a large amount of F-actin in the apical region of the sperm head. The incubation of ultrathin sections of sperm embedded in Lowicryl K4M with a phalloidin–gold complex allowed a more precise localization of F-actin in the amorphous part of the acrosome and in the cytoplasmic region between acrosome and nucleus; F-actin is also present in the thin cytoplasmic layer between plasma membrane and nuclear envelope at the apical portion of the nucleus. Although the sperm was always found completely devoid of motility, the discovery of the presence of an actin cytoskeleton leads us to hypothesize a possible acquisition of motility by the sperm at the time of its interaction with the female gamete. Such a hypothesis is supported by what is known for ostracods whose aflagellate sperm implement a type of amoeboid movement only at the time of their interaction with the female gamete.

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

The N-terminal proline-rich domain of γ-zein (Zera) plays an important role in protein body (PB) formation not only in the original host (maize seeds) but in a broad spectrum of eukaryotic cells. However, the elements within the Zera sequence that are involved in the biogenesis of PBs have not been clearly identified. Here, we focused on amino acid sequence motifs that could be involved in Zera oligomerization, leading to PB-like structures in Nicotiana benthamiana leaves. By using fusions of Zera with fluorescent proteins, we found that the lack of the repeat region (PPPVHL)(8) of Zera resulted in the secretion of the fusion protein but that this repeat by itself did not form PBs. Although the repeat region containing eight units was the most efficient for Zera self-assembly, shorter repeats of 4-6 units still formed small multimers. Based on site-directed mutagenesis of Zera cysteine residues and analysis of multimer formation, we conclude that the two N-terminal Cys residues of Zera (Cys(7) and Cys(9)) are critical for oligomerization. Immunoelectron microscopy and confocal studies on PB development over time revealed that early, small, Zera-derived oligomers were sequestered in buds along the rough ER and that the mature size of the PBs could be attained by both cross-linking of preformed multimers and the incorporation of new chains of Zera fusions synthesized by active membrane-bound ribosomes. Based on these results and on the behavior of the Zera structure determined by molecular dynamics simulation studies, we propose a model of Zera-induced PB biogenesis.

Immunolocalization of humanin in human sperm and testis

We have discovered, by immunocytochemistry and immunoelectronmicroscopy, that humanin (HN) is expressed in human ejaculated sperm and testis. In sperm, the HN immunolabeling pattern depends on sperm morphology; in particular, HN is mainly localized in the midpiece of sperm in semen samples with normal morphology and in cytoplasmic residues and entire tail in those with abnormal morphology. We also found HN in the cytoplasm and nucleus of spermatocytes and spermatids and in experimentally uncoiled chromatin of mature ejaculated sperm. Because it has been established that HN has antiapoptotic properties, it is reasonably hypothesized that HN may play an important role in preventing apoptosis in human sperm and testis. Thus, the examination of the HN localization in normal and abnormal sperm could be proposed as an auxiliary test to better define sperm quality.

Early events of secretory granule formation in the rat parotid acinar cell under the influence of isoproterenol. An ultrastructural and lectin cytochemical study.

The events involved in the maturation process of acinar secretory granules of rat parotid gland were investigated ultrastructurally and cytochemically by using a battery of four lectins [Triticum vulgaris agglutinin (WGA), Ulex europaeus agglutinin I (UEA-I), Glycine max agglutinin (SBA), Arachys hypogaea agglutinin (PNA)]. In order to facilitate the study, parotid glands were chronically stimulated with isoproterenol to induce secretion. Specimens were embedded in the Lowicryl K4M resin. The trans-Golgi network (TGN) derived secretory granules, which we refer to as immature secretory granules, were found to be intermediate structures in the biogenesis process of the secretory granules in the rat parotid acinar cell. These early structures do not seem to be the immediate precursor of the mature secretory granules: in fact, a subsequent interaction process between these early immature granule forms and TGN elements seems to occur, leading, finally, to the mature granules. These findings could explain the origin of the polymorphic subpopulations of the secretory granules in the normal acinar cells of the rat parotid gland. The lectin staining patterns were characteristic of each lectin. Immature and mature secretory granules were labelled with WGA, SBA, PNA, and lightly with UEA-I. Cis and intermediate cisternae of the Golgi apparatus were labelled with WGA, and trans cisternae with WGA and SBA.

CGI-58 Is an α/β-Hydrolase within Lipid Transporting Lamellar Granules of Differentiated Keratinocytes

CGI-58 is the causative molecule underlying Dorfman-Chanarin syndrome, a neutral lipid storage disease exhibiting apparent clinical features of ichthyosis. CGI-58, associated with triacylglycerol hydrolysis, has an alpha/beta-hydrolase fold and is also known as the alpha/beta-hydrolase domain-containing protein 5. The purpose of this study was to elucidate the function of CGI-58 and the pathogenic mechanisms of ichthyosis in Dorfman-Chanarin syndrome. Using an anti-CGI-58 antibody, we found CGI-58 to be expressed in the upper epidermis, predominantly in the granular layer cells, as well as in neurons and hepatocytes. Immunoelectron microscopy revealed that CGI-58 was also localized to the lamellar granules (LGs), which are lipid transport and secretion granules found in keratinocytes. CGI-58 expression was markedly reduced in the epidermis of patients with harlequin ichthyosis, demonstrating defective LG formation. In cultured keratinocytes, CGI-58 expression was mildly up-regulated under high Ca(2+) conditions and markedly up-regulated in three-dimensional, organotypic cultures. In the developing human epidermis, CGI-58 immunostaining was observed at an estimated gestational age of 49 days, and CGI-58 mRNA expression was up-regulated concomitantly with both epidermal stratification and keratinocyte differentiation. CGI-58 knockdown reduced expression of keratinocyte differentiation/keratinization markers in cultured human keratinocytes. Our results indicate that CGI-58 is expressed and packaged into LGs during keratinization and likely plays crucial role(s) in keratinocyte differentiation and LG lipid metabolism, contributing to skin lipid barrier formation.

FSCB, a Novel Protein Kinase A-phosphorylated Calcium-binding Protein, Is a CABYR-binding Partner Involved in Late Steps of Fibrous Sheath Biogenesis

We report characterization of a novel testis- and sperm-specific protein, FSCB (fibrous sheath CABYR binding), that is expressed post-meiotically and localized in mouse sperm flagella. FSCB was identified as a binding partner of CABYR, a calcium-binding protein that is tyrosine-phosphorylated during capacitation. Orthologous genes of FSCB are present in other mammals, including rat and human, and conserved motifs in FSCB include PXXP, proline-rich and extensin-like regions. FSCB is phosphorylated by protein kinase A as shown by in vitro phosphorylation assay and also by determining phosphorylation sites in native FSCB from mouse sperm. Calcium overlay assay showed that FSCB is a calcium-binding protein from sperm. FSCB is a post meiotic protein first expressed at step 11 of mouse spermatogenesis in the elongating spermatids, and it subsequently incorporates into the flagellar principal piece of the sperm. Ultrastructurally, FSCB localized to a cortical layer of intermediate electron density at the surface of the ribs and longitudinal columns of the fibrous sheath. Due to its temporal appearance during spermiogenesis and location at the cortex of the fibrous sheath, FSCB is postulated to be involved in the later stages of fibrous sheath assembly.

In situ molecular identification of the Ntf4 MAPK expression sites in maturing and germinating pollen

MAPKs (mitogen-activated protein kinases) are involved in the transduction of different signals in eukaryotes. They regulate different processes, such as differentiation, proliferation and stress response. MAPKs act through the phosphorylation cascade, being the last element that phosphorylates the final effector of the cell response. They are activated when their threonine and tyrosine residues are phosphorylated. Ntf4, a MAPK with a molecular mass of 45 kDa, has been reported to be expressed in pollen and seeds. Biochemical studies have indicated that the expression and the activation of Ntf4 is regulated during pollen maturation, although an increase of the activation is observed when the pollen is hydrated, just at the beginning of the germination. However, nothing is known about its subcellular localization. In the present study, the in situ expression and subcellular localization of Ntf4 have been analysed during the tobacco pollen developmental pathway. Cryosections, freeze-substitution and cryo-embedding in Lowicryl K4M were used as processing techniques for subsequent immunofluorescence, immunogold labelling and in situ hybridization assays. During pollen maturation, Ntf4 showed an increase in expression, as demonstrated by in situ hybridization, and specific subcellular distributions. We found that the protein was expressed from mid bicellular pollen stage until the pollen was mature. In germinating pollen, the protein increased after the initiation of germination. Translocation of the protein to the nucleus was found at specific stages; the presence of Ntf4 in the nucleus was found in the last stage of the pollen maturation and in germinating pollen. Double immunofluorescence and immunogold labelling with anti-Ntf4 (AbC4) and anti-P-MAPK (phosphorylated MAPK) antibodies revealed the co-localization of both epitopes in the nucleus at late developmental stages. The temporal and spatial pattern of the expression sites of Ntf4 has been characterized during pollen development, indicating that Ntf4 is a 'late gene' that is upregulated during maturation and germination, with a possible role in the gametophytic function. The translocation of the Ntf4 protein from the cytoplasm to the nucleus at late pollen developmental stages, and its co-localization with the P-MAPK epitope in several nuclear sites, indicates a relationship between the Ntf4 nuclear translocation and its active state.

Platinum blue as an alternative to uranyl acetate for staining in transmission electron microscopy

This paper introduces an aqueous solution of platinum blue (Pt-blue) as an alternative to uranyl acetate (UA) for staining in transmission electron microscopy (TEM). Pt-blue was prepared from a reaction of cis-dichlorodiamine-platinum (II) (cis-platin) with thymidine. When Pt-blue was dried on a microgrid and observed by TEM it showed a uniform appearance with tiny particles less than 1 nm in diameter. The effect of Pt-blue as an electron stain was then examined not only for positive staining of conventional ultrathin resin sections and counterstaining of post-embedding immuno-electron microscopy but also for negative staining. In ultrathin sections of the rat liver and renal glomerulus, Pt-blue provided good contrast images, especially in double staining combined with a lead stain (Pb). Almost all cell organelles were clearly observed with high contrast in these sections. Glycogen granules in the hepatic parenchymal cells were particularly electron dense in Pt-blue stained sections compared with those treated with UA. In longitudinal and transverse sections of budding influenza A viruses, a specific arrangement of rod-like structures, which correspond to the ribonucleoprotein complexes, was clearly shown in each virion stained with Pt-blue and Pb. When post-embedding immunoelectron microscopy was performed in ultrathin sections of HeLa cells embedded in Lowicryl K4M, the localization of Ki-67 protein was sufficiently detected even after Pt-blue and Pb staining. The present study also revealed that Pt-blue could be used for the negative staining of E. coli, allowing the visualization of a flagellum. These findings indicate that Pt-blue is a useful, safe, and easily obtainable electron stain that is an alternative to UA for TEM preparations.

Menkes protein localization in rat parotid acinar cells

The aim was to study the subcellular localization of the Menkes protein (MNK; ATP7A) in the rat parotid acinar cell. MNK protein is a copper transporting P-type ATPase whose absence or dysfunction causes a fatal neurodegenerative disorder, MNK disease. Rat parotid glands were fixed and low-temperature embedded in Lowicryl K4M resin, and ultrathin sections were prepared for immunocytochemical analysis. Immunolocalization of MNK was demonstrated mainly over the trans Golgi network (TGN) area. Immature and mature secretory granules were also labelled, indicating that MNK protein could be involved here in copper secretion from acinar cells into saliva, consistent with a proposed cariostatic role for copper.

Immunohistochemical Detection of the Cytoskeletal Components in Gastric Parietal Cells

Gastric parietal cells are characterized by intracellular canaliculi with many microvilli and tubulovesicles. These membranous structures are continuous and change in volume according to gastric acid secreting or resting state. Morphological changes are induced by cytoskeletal actin. Actin filaments are anchored to the cell membrane via ezrin. This study aimed to ascertain whether morphological changes were related to the nature of ezrin molecules, which were either phosphorylated or non-phosphorylated. Rat stomach tissues were rapid-frozen followed by freeze-substitution and embedding in paraffin for light microscopy. Also, the specimens were high pressure-frozen, freeze-substituted and embedded in Epon 812 or Lowicryl K4M for electron microscopy. Sections from paraffin or Lowicryl K4M were stained with anti-H+·K+-ATPase antibodies, anti-phosphorylated or non-phosphorylated ezrin antibodies, and anti-actin antibody. The H+·K+-ATPase was distributed on the intracellular canaliculi and tubulovesicles. The intracellular canaliculi were labeled with anti-phosphorylated ezrin antibodies in fed (gastric acid secreting state) rats. These were stained weakly in starved (gastric acid resting state) rats. Anti-nonphosphorylated ezrin antibodies hardly stained these organelles in either state. A possible explanation is that the phosphorylation of ezrin molecules is related to the reciprocal membranous transformation between intracellular canaliculi and tubulovesicles.

Hereditary 'white nails': a genetic and structural study.

Hereditary subtotal leuconychia is a rare nail disease. The gene(s) underlying this phenotype is (are) not known. Immunohistochemical and ultrastructural studies of nails are performed infrequently. To perform genetic linkage analysis and to assess ultrastructure and soft/hard keratin expression in hereditary white nails. We have analysed microscopically and ultrastructurally the white nails of a patient from a family in which the trait is inherited in an autosomal dominant manner as an isolated symptom. No skin lesions or hair abnormalities could be detected. Genetic linkage studies were performed on DNA samples obtained from several members of the affected family. A longitudinal surgical biopsy of the nail from a great toe was split in two parts. One part was fixed in formalin and processed for histopathology. Another part was further subdivided and embedded either in Epon, following fixation in 2% glutaraldehyde, or in Lowicryl K4M, after fixation in 3% paraformaldehyde. Dewaxed nail sections and Lowicryl ultrathin sections were also stained with various antikeratin antibodies. Genetic linkage studies of the family pointed to the disease gene mapping to the chromosomal 12q13 region. Genes mapping within this chromosomal region include the genes coding for type II (basic) cytokeratins and hard keratins. The nail matrix presented an abnormal hypergranulosis. The upper part of the nail plate, originating from the proximal nail matrix, had a nonhomogeneous lamellar appearance, with numerous intracellular 'lipidic' vacuoles and 'empty' spaces separating keratin filament bundles. These cells were progressively shed at the nail surface. The cell loss was compensated by hyperproliferation of the distal matrix and of the nail bed keratinocytes, with persistent marked parakeratosis and loose arrangement of keratin bundles. The distal matrix and the nail bed contributed equally to formation of the lower plate. This presented the characteristics of a tissue composed of soft keratins. Accordingly, there was virtually no labelling with the Hb1 antibody to a basic hard keratin in the white nail, whereas the labelling with AE3 antibody to all type II keratins and with KL1 recognizing suprabasal soft keratins was normal or even enhanced. Genetic linkage indicates that the gene defect underlying the leuconychia in the family studied resides on chromosome 12q13. As the type II keratins map within this chromosomal interval, it is possible that a mutation in one of these keratin genes may be a cause of the hereditary leuconychia. The white appearance of nails in this disease seems to be due to an abnormal keratinization of cells originating from the proximal nail matrix, leading to the presence of abundant intracellular vacuoles and to a lesser compactness of keratins.

Antigen retrieval trial for post-embedding immunoelectron microscopy by heating with several unmasking solutions.

A novel antigen retrieval procedure was carried out in the post-embedding immunogold electron microscopy method to improve the stainability of the samples. This was done by weakly fixing cultured Helicobacter pylori (ATCC43504) and embedding in Lowicryl K4M. Before staining with the anti-H. pylori antibody, the ultrathin sections were mounted on a nickel grid and heated at 121C for 15 min, 99C for 40 min, and 65C for 24 hr in distilled water, 0.1 M phosphate buffer (pH 7.4), 0.01 M EDTA (pH 7.2), 0.05 M Tris buffer (pH 10.0), 0.8 M urea (pH 7.2), 0.01 M citric acid (pH 6.0), or a commercially available target unmasking fluid (S1699; pH 6.0). Antigen retrieval in the Tris buffer solution generally showed better stainability than the classical post-embedding method without any antigen retrieval. At 65C for 24 hr, better stainability of the ultrasections was observed for each of the solutions used except for the phosphate buffer compared to the control. We suggest that the antigen retrieval method should be applied for routine use even by in post-embedding immunogold electron microscopy.

Immunomapping of EBA sera to multiple epitopes on collagen VII: further evidence that anchoring fibrils originate and terminate in the lamina densa.

Epidermolysis bullosa acquisita (EBA) is an autoimmune blistering disease with circulating antibodies to type VII collagen, a major component of anchoring fibrils located at the dermal-epidermal junction. The purpose of this study was to further confirm the ultrastructural organisation of anchoring fibrils and to assess the relationship between the clinical phenotype of EBA and target site of their autoantibodies on anchoring fibrils. We studied the ultrastructural binding site of circulating autoantibodies from two patients with atypical clinical features who predominantly presented with oral lesions, and compared this with two patients with clinically typical forms of EBA. Immunoblotting of whole dermal extracts showed labelling of 290-kDa bands consistent with that of type VII collagen as well as the non-collagenous (NC-1) domain fusion protein in three out of four patients' sera. Postembedding immunoelectron microscopy (IEM) using Lowicryl K11M embedded normal human skin and patients' sera demonstrated the majority of labelling within the lamina densa, not below the lamina densa. We conclude that EBA autoantibodies in these patient's sera bind to the NC-1 domain of collagen VII situated in the lamina densa of the epidermal basement membrane, regardless of the EBA clinical phenotype. This confirms the previous notion that anchoring fibrils originate and terminate in the lamina densa.

Cytochemistry of gastric parietal cells with high-pressure freezing followed by freeze-substitution.

Gastric parietal cells were examined for changes in their ultrastructure and distribution of the proton pump during feeding and fasting states in rats. The fundic glands from rats fed ad libitum or fasted with free access to water were cryofixed using high-pressure freezing followed by freeze-substitution in acetone containing osmium or acrolein and then embedded in Epon 812 or Lowicryl K4M resin, respectively. Excellent ultrastructural preservation was achieved. During the feeding state, intracellular canaliculi and numerous microvilli were well developed, while tubulovesicles were poorly developed. In contrast, during the fasting state, the microvilli in the narrowed space of the intracellular canaliculi were tightly packed and the tubulovesicles were enlarged. Ultrathin sections were immunostained with antibodies against the alpha- and beta-subunits of the proton pump, H+ x K(+)-ATPase, using the immunogold method. The labelling was strong and clearly localized in comparison with that obtained using the conventional chemical-fixation method. Each subunit was localized on the membrane of the microvilli, intracellular canaliculi and tubulovesicles. The distribution of subunit proteins varied between the two states. During ad libitum feeding, the immunolabelling was localized strongly on the membranes of the microvilli and intracellular canaliculi. In contrast, the labelling was strong on the tubulovesicle membrane in the fasting state. The results obtained with each anti-subunit antibody by H+ x K(+)-ATPase immunostaining revealed differences in distribution and labelling density between the feeding and fasting states.

Electron microscopic immunogold localization of salivary mucins MG1 and MG2 in human submandibular and sublingual glands.

The human salivary mucins MG1 and MG2 are well characterized biochemically and functionally. However, there is disagreement regarding their cellular and glandular sources. The aim of this study was to define the localization and distribution of these two mucins in human salivary glands using a postembedding immunogold labeling method. Normal salivary glands obtained at surgery were fixed in 3% paraformaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M or LR Gold resin. Thin sections were labeled with rabbit antibodies to MG1 or to an N-terminal synthetic peptide of MG2, followed by gold-labeled goat anti-rabbit IgG. The granules of all mucous cells of the submandibular and sublingual glands were intensely reactive with anti-MG1. No reaction was detected in serous cells. With anti-MG2, the granules of both mucous and serous cells showed reactivity. The labeling was variable in both cell types, with mucous cells exhibiting a stronger reaction in some glands and serous cells in others. In serous granules, the electron-lucent regions were more reactive than the dense cores. Intercalated duct cells near the acini displayed both MG1 and MG2 reactivity in their apical granules. In addition, the basal and lateral membranes of intercalated duct cells were labeled with anti-MG2. These results confirm those of earlier studies on MG1 localization in mucous cells and suggest that MG2 is produced by both mucous and serous cells. They also indicate differences in protein expression patterns among salivary serous cells.

Effect of deproteination on bone mineral morphology: implications for biomaterials and aging

Bone mineral morphology is altered by processing and this is rarely considered when preparing bone as a bioimplant material. To examine the degree of transformation, a commercial, coarsely particulate bone mineral biomaterial produced by prolonged deproteination, defatting, dehydration, and heating (donor material) was compared with similar particles of human bone (recipient material) prepared optimally by low-temperature milling. The two powders were freeze-substituted and embedded without thawing in Lowicryl K4M before sectioning for transmission electron microscopy (TEM) (other aliquots were processed by traditional TEM methods). To maximize resolution, electron micrographs were image-enhanced by digitization and printed as negatives using a Polaroid Sprint Scan 45. In addition to their morphology, the particles were examined for antigenicity ( specific by reference to fluorescein isothiocyanate [FITC]-conjugated fibronectin, and nonspecific by reference to general FITC-conjugated immunoglobulins). Results showed that the optimally prepared human bone fragments stained discretely for fibronectin with negligible background autofluorescence. In contrast, the bioimplant fragments stained extensively with this and any other FITC-conjugated antibody and, unlike fresh bone, it also autofluoresced a uniform yellow. This difference was also expressed structurally and, although the bioimplant mineral consisted of rhomboidal plates up to 200 nm across and 10 nm thick, the optimally prepared bone mineral was composed of numerous clusters of 5-nm-wide sinuous calcified filaments of variable density and indeterminate length (which became straight needles 50 nm long and 5 nm thick following traditional chemical TEM fixation/staining). It was concluded that the inorganic phase of bone is both morphologically and immunologically transmutable and that, in biomaterials, the transformation is apparently so great that a broad indigenous antigenicity is unmasked, increasing the likelihood of resorption or rejection. This marked change may also provide preliminary insight into a more modest natural aging phenomenon with the localized lateral fusion of calcified filaments into less flexible, more immunologically reactive fenestrated plates.

Cytochemical investigation of gastric gland component cells with high-pressure freezing followed by freeze-substitution and hydrophilic resin embedding.

Gastric gland component cells were electron-microscopically and immunoelectronmicroscopically examined with high-pressure freezing followed by freeze substitution and a low-temperature embedding resin method and compared to that of the conventional chemical-fixation method. The rat gastric gland was high-pressure frozen, freeze-substituted with acetone-containing osmium or acrolein, and embedded in Epon 812 or Lowicryl K4M, respectively. Using the high-pressure freezing method, the vitreous freezing range reached the depth of 150 microns from the surface. The ultrathin sections from both procedures embedding in Epon 812 and Lowicryl K4M were doubly stained with uranyl acetate and lead acetate, and histochemically or immunohistochemically stained, respectively. In comparison to the conventional chemical fixation method, excellent results were obtained with respect to ultrastructural preservation. The stainings performed in this experiment included periodic acid-thiocarbohydrazide-silver proteinate staining, cationic colloidal cold at pH 2.5 staining, Helix pomatia lectin-staining, anti-alpha or -beta subunit antibodies of H+K(+)-ATPase immunostaining and pepsinogen immunostaining. The staining intensity of those was stronger than that of the conventional immersion-chemical fixation method. In addition to these results, the labels also showed good specific localization. In this paper, we provide a description of the high-pressure freezing followed by freeze substitution and low-temperature embedding resin method compared to the conventional chemical-fixation method. Our results suggest that this method is a suitable tool for ultrastructural and histochemical/immunohistochemical studies at high resolution.

Fluid dynamics of the excretory flow of zymogenic and mucin contents in rat gastric gland processed by high-pressure freezing/freeze substitution.

The high-pressure freezing/freeze substitution technique followed by Lowicryl K4M embedding provided an excellent ultrastructure and retention of antigenicity of rat gastric glands as well as the intraluminal fluid contents. By taking this advantage, we histochemically investigated the excretory flow of the zymogenic and mucin contents in rat gastric glandular lumen at the ultrastructural level. The combination of KMnO(4)-UA/Pb staining for zymogenic contents and Griffonia simplicifolia agglutinin-II (GSA-II) labeling for mucous neck cell (MNC) mucin distinguished the exocytosed zymogenic contents from the MNC mucin in the glandular lumen. Interestingly, at the base and neck regions, the zymogenic contents showed a droplet-like appearance, forming a distinct interface with the MNC mucin. At the pit region, the GSA-II labeling demonstrated restricted paths, designated as MNC mucous channels, which flowed into the surface mucous gel layer. It should be noted that the interface between exocytosed zymogenic contents and MNC mucin disappeared, and that the zymogenic contents merged into the MNC mucous channels. At the top pit region, the surface mucous gel layer showed laminated arrays of three types of gastric mucins. On the basis of these ultrastructural findings, we propose a model of the excretory flow in rat gastric gland.

Immunocytochemical demonstration of the secretory dynamics of zymogenic contents in rat gastric gland processed by high-pressure freezing/freeze substitution, with special references to phospholipase A(2) and phospholipase Cgamma1.

High-pressure freezing/freeze substitution followed by Lowicryl K4M embedding provided an excellent morphology and antigenicity of the gastric glands, as well as the intraluminal fluid contents. Taking advantage of this, we histochemically investigated the secretory dynamics of the zymogenic contents in rat gastric gland, with special references to phospholipase A(2) (PLA(2)) and phospholipase Cgamma1 (PLCgamma1). The combination of immunogold labeling and KMnO4-uranyl acetate-lead citrate staining for zymogenic contents clearly demonstrated the rapid diffusion of PLA(2) molecules from the exocytosed zymogenic contents into the mucinous contents in gastric glandular lumens. In contrast, the exocytosed PLCgamma1 molecules remained within the zymogenic contents in the glandular lumens. These findings indicated the distinction between the exocytosed PLA(2) and PLCgamma1 in their diffusion rate. In addition, the mucinous contents surrounding the exocytosed zymogenic contents were intensely labeled with Griffonia simplicifolia II lectin which specifically recognizes the mucin of mucous neck cells. Interestingly, some of the PLA(2) immunolabeling on the mucinous contents was associated with the apical membranes of gastric epithelial cells, especially that of parietal cells. The secretory dynamics of the zymogenic contents in rat gastric glands, including their interaction with the mucinous contents are discussed.