Influence of two different fixatives on the identification of plasma cells in human rectal mucosa

Plasma cells in sections of bisected human rectal biopsy specimens, fixed in two alternative fixatives, were enumerated after staining by an indirect immunoperoxidase procedure intended to demonstrate immunoglobulin-containing cells. The counts of immunoperoxidase-positive plasma cells were significantly higher after fixation in formol sublimate than after fixation in formol saline. Formol sublimate appears to be a more reliable fixative than formol saline for specimens of rectal mucosa in which quantitation of plasma cells, stained for intracellular immunoglobulin by an immunoperoxidase technique, is intended.     

Chemical and Spectrometric Evaluation of Glycogen After Routine Histological Fixatives

A detailed comparison of fixatives used for the demonstration of glycogen has been based on chemical assay and microspectrophotometry. Rat liver containing known amounts of glycogen was fixed in formol alcohol, Rossman's fluid, 10% neutral formalin, Bouin, Helly, SUSA, and Zenker's fluid at 4 C and 18 C. Chemical assay was carried out before and after fixation and paraffin sections were prepared from the fixed material. Sections were stained with PAS and the silver methenamine method. Visual examination was carried out with a comparison microscope and quantitative estimations on PAS-stained sections were performed by scanning microspectrophotometry. The histochemical methods were compared with the chemical results obtained from the same tissue and a reasonable degree of correlation between the sets of results was observed. Cold formol alcohol and cold Rossman's fluid preserved the most glycogen and Zenker and SUSA fixation preserved the least. Cold formol alcohol was the only fixative that preserved threshold values of glycogen i.e. 0.3% and the silver methenamine method is recommended for the demonstration of these small amounts.     

Human ferritin: effects of antigen source and fixation on leucocyte staining by immunoperoxidase technique

The localization of ferritin was studied in peripheral blood cells and variously fixed tissues with the antibodies against ferritins isolated from human heart and spleen. The unlabelled antibody enzyme method (PAP) was used to detect the binding sites of antibodies. In peripheral blood cell smears both antisera gave rise to strong staining of polymorphonuclear (PMN) cell cytoplasm, whereas the monocytes stained relatively weakly. There were no staining differences between the two antisera. In human spleen sections the spleen ferritin antiserum stained the PMN cells and sinusoidal lining cells, whereas the heart ferritin antiserum stained only PMN cells. Neither of the two antisera stained monocytes in the spleen sections. This finding was observed in specimens fixed in Bouin's fixative, Baker's fixative and neutral formalin. However, the immunoreactivity of ferritin was totally destroyed by some other fixatives (Carnoy's fixative, formol sucrose and glutaraldehyde). These results suggest that ferritin is more readily released from monocytes than from PMN cells, and that mature spleen macrophages contain antigenic determinants of ferritin that are recognized only by anti-spleen ferritin antiserum.     

Staining Sulfated Mucopolysaccharides in Sections by Means of Acriflavine

Acriflavine gave insoluble salts with sulfated esters. Frozen or paraffin sections (fixed in 10% formol or Carnoy's solution) were stained in M/20 acriflavine solution and excess dye was rinsed in 95% alcohol. Then nuclei were stained with Meyer's haemalum. Thereafter the sections were washed in water, dehydrated in alcohol, cleared in xylene and mounted in balsam. Sulfated esters in the tissue sections were colored yellow or orange-yellow, generally more densely in frozen than in paraffin sections.     

In situ localization of amylase mRNA and protein. An investigation of amylase gene activity in normal human parotid gland

The distribution of human salivary amylase mRNA was studied by in situ hybridization to a [32P]-labeled amylase cDNA probe. Amylase mRNA was localized to the apical portion of acinar cells in frozen sections of human parotid salivary gland. No hybridization was noted in ductal cells, skeletal muscle, or in connective tissue. These results were consistent with immunohistochemical localization of amylase. The technique of in situ hybridization was modified to permit localization of amylase mRNA in variously fixed, paraffin-embedded parotid glands. Although the hybridization signal decreased with all fixatives, the pattern of localization paralleled that obtained with frozen sections. No advantage was noted in fixation with ethanol-acetic acid or Bouin solution over routine fixation with formalin. These results have important implications for researchers interested in studies of gene expression. We have demonstrated that routinely fixed paraffin blocks of human tissue can be used for cellular localization of specific mRNA. In coordination with immunocytochemistry, in situ hybridization offers a powerful tool for studies of mRNA and protein expression in individual cells.     

Human ferritin: Effects of antigen source and fixation on leucocyte staining by immunoperoxidase technique

Summary The localization of ferritin was studied in peripheral blood cells and variously fixed tissues with the antibodies against ferritins isolated from human heart and spleen. The unlabelled antibody enzyme method (PAP) was used to detect the binding sites of antibodies. In peripheral blood cell smears both antisera gave rise to strong staining of polymorphonuclear (PMN) cell cytoplasm, whereas the monocytes stained relatively weakly. There were no staining differences between the two antisera. In human spleen sections the spleen ferritin antiserum stained the PMN cells and sinusoidal lining cells, whereas the heart ferritin antiserum stained only PMN cells. Neither of the two antisera stained monocytes in the spleen sections. This finding was observed in specimens fixed in Bouin's fixative, Baker's fixative and neutral formalin. However, the immunoreactivity of ferritin was totally destroyed by some other fixatives (Carnoy's fixative, formol sucrose and glutaraldehyde). These results suggest that ferritin is more readily released from monocytes than from PMN cells, and that mature spleen macrophages contain antigenic determinants of ferritin that are recognized only by anti-spleen ferritin antiserum.Supported by the Sigrid Jusélius Foundation and Finska Läkaresällskapet. Y.T.K. is a recipient of a grant for post-doctoral fellowship of Helsinki University     

Immunoglobulin (Ig)-containing plasma cells in the Harderian gland in broiler and native chickens of Bangladesh

The distribution and frequency of immunoglobulin (Ig)-containing plasma cells, their variations due to sex, and the mode of secretion of Ig cells into the duct system of the Harderian gland was investigated in broiler and native chickens of both sexes in Bangladesh. The Harderian gland is covered by a capsule, and the connective tissue septa divide the gland into numerous unequal-sized numerous lobes and lobules. The Ig-containing plasma cells were located in the interstitial space, interacinar space, apical part of the lobule, and lumina of the lobules of the Harderian gland in both broiler and native chickens. The population of these Ig-containing plasma cells varied in between broiler and native chickens, and also between male and female broiler and native chickens. In the broiler, the number of IgM-containing plasma cells was higher; in contrast, in the native chickens, the population of IgA-containing plasma cells was larger. In the broiler, there were more IgA- and IgG-containing plasma cells in the male; in contrast, there were more IgM-containing plasma cells in female. In native chickens the frequency of IgA-containing plasma cells was greater in the female than male. When the data for broiler and native birds were compared, it was found that there were significantly more IgA- and IgG-containing plasma cells in the native male and female chickens than in the broiler males and females. The secretory Igs were located in the lumina of acini and the duct system of the Harderian gland. In the present study Ig-containing plasma cells were observed to be released in the lumina of the lobules of Harderian gland by the breakdown of acinar tissues in broilers, and by holocrine mode of secretion in the native chicken. These results suggested that the Harderian gland, even though it is not a lymphoid organ as a whole, but acts as an immunopotent organ in chickens, and that the gland in native chicken contains more Ig-containing plasma cells due to their scavenging.     

Polyester wax embedding and sectioning technique for immunohistochemistry

We have developed a method useful for immunohistochemical studies by combining tissue fixation with buffered neutral formalin and polyester wax embedding. Buffered neutral formalin fixation preserves cell and tissue fine structure, and also the antigenicity of unstable enzymes. Polyester wax embedding makes possible thin serial sections of various tissues and preserves antigenicities for at least 6 months. We have demonstrated using this technique the localization of alpha-amylase in mouse salivary gland, parietal-cell specific antigen in mouse glandular stomach, and DNA polymerase alpha and beta in chick tissue.     

Use of Gallocyanin as a Myelin Stain for Brain and Spinal Cord

Tissue fixed in 10% formalin, formol saline, CaCO₃ or phosphate buffer neutralized formalin, Baker's formol calcium, Cajal's formol ammonium bromide, formalin-95% ethanol 1:9, formalin-methanol 1:9, Lillie's methanol-chloroform or Salthouse's formol cetyltrimethylammonium bromide was dehydrated and embedded in paraffin. Sections were attached to slides with either albumen or gelatine adhesive and processed throughout at room temperature of 22-25 C. Mordanting 30-60 min in 1% iron alum was followed by a 10 min wash in 4 changes of distilled water. Myelin was stained in a gallocyanin self-differentiating solution for 1-2.5 hr; thick sections requiring the longer time. The staining solution (pH approximately 7.4) consisted of Na₂CO₃, 90 mg; distilled water, 100 ml; gallocyanin, 250 mg; and ethanol, 5 ml. The ethanol was added to this mixture last, and after the other ingredients had been boiled and then cooled to room temperature. After a staining and thorough washing, Nissl granules were stained for 5-10 min in a solution consisting of: 0.1 M acetic acid, 60 ml; 0.1 M sodium acetate, 40 ml; methyl green, 500 mg. Washing, dehydration, clearing and mounting completed the process. Myelin sheaths were stained dark violet; neuronal nuclei, light green with dark granules of chromatin; nucleoli of motor cells and erythrocytes, dark violet; cytoplasm, green with dark green Nissl granules. The simple and reliable method can be adapted easily for use with automatic tissue processors.     

Characteristics of human IgA and IgM genes used by plasma cells in the salivary gland resemble those used in duodenum but not those used in the spleen

Immunologically, the parotid salivary gland is an effector site that secretes large quantities of polyspecific Abs into the saliva, mainly of the IgA isotype. It is considered to be part of the common mucosal immune system but the inductive site for the Ab-producing cells of the salivary gland has not yet been clearly identified. The origin and diversity of cells of B lineage can be investigated by analyzing their Ig heavy chain genes (IgH). We have obtained sequences of IgM and IgA VH4-34 genes from plasma cells in human salivary gland, duodenal lamina propria, and splenic red pulp. Related sequences were found in different areas sampled within each tissue studied, indicating that the plasma cells carrying these genes are widespread with limited diversity. Examples of related IgH genes that are isotype switched were also seen in the salivary gland. The genes from plasma cells of the salivary gland were highly mutated, as were duodenal plasma cell sequences. The level of mutation was significantly higher than that seen in splenic plasma cell sequences. Analysis of CDR3 regions showed that the sequences from salivary gland had significantly smaller CDR3 regions than sequences from spleen, due to differences in number and type of DH regions used. Sequences from duodenum also had smaller CDR3 regions. Therefore, plasma cells from human duodenum and salivary gland showed characteristics that differed from those of human splenic plasma cells.     

Functional morphology of the salivary gland of the snail, Helix pomatia: a histochemical and immunocytochemical study

Functional morphology of Helix pomatia salivary gland cells was studied at light microscopic level by using different histochemical methods. Three cell types could be demonstrated in the salivary gland: mucocytes, granular and vacuolated cells. The distribution and the number of the different cell types were different in active and inactive snails. In active feeding animals, dilatated interlobular salivary ducts were observed, which were never present in inactive ones. In active animals an additional cell type, the cystic cell could also be observed. Periodic acid Schiff staining revealed both mucuos and serous elements in the salivary gland. Furthermore, hematoxyline-eosin staining indicated the occurrence of a cell layer with high mitotic activity in the acini. Applying immunohistochemical methods with monoclonal mouse anti-human Ki-67 clone, B56 and polyclonal rabbit anti-human Ki-67 antibodies, we also were able to demonstrate the occurrence of dividing cells in the salivary gland. Analysis of 1-2 microm semi-thin Araldite sections stained with toluidine-blue showed that the saliva can be released, in addition to possible exocytosis, by the lysis of cystic cells. Using an apoptosis kit, we could also establish that this process was due to rather an apoptotic than a necrotic mechanism. In the salivary gland of active snails, where an intensive salivation takes place, significantly more apoptotic cells occurred, if compared to that of inactive animals. It is suggested that programmed cell death may also be involved in the saliva release.     

Human kallikrein 13 expression in salivary gland tumors

The human kallikrein 13 protein (hK13) is expressed in many normal tissues. Petraki et al have previously described presence of hK13 in salivary gland tissue, localized to duct epithelia and some acinar cells. The aim of this study was to determine whether hK13 is expressed in salivary gland tissues and salivary gland tumors (both benign and malignant), in order to compare normal with tumor tissues. Pleomorphic adenomas (PA), adenoid cystic carcinomas (ACC), polymorphous low grade adenocarcinomas (PLGA), acinic cell carcinomas (ACI), mucoepidermoid carcinomas (MEC) and adenocarcinomas not otherwise specified (ANOS) of both minor and major salivary glands were examined. The results of this study indicate that most salivary gland tumors show high levels of expression of hK13. Overall, staining in PA was significantly less than that seen in normal salivary gland tissue. PLGA, ACC and ANOS each stained significantly more than normal salivary gland tissue while MEC and ACI did not. Ductal cells and cells lining duct-like structures showed a higher intensity of staining than non-ductal cells in most tumors. Tumors which exhibited only non-ductal cells also exhibited cytoplasmic staining. In conclusion, we demonstrate the high expression of hK13 in several common salivary gland tumors.     

Anatomy of the major lacrimal gland of rhesus monkeys (Macaca mulatta)

The major lacrimal gland of rhesus monkeys is impalpable within the fatty connective tissue of the upper lateral quadrant of the orbit. Acini of the lacrimal glands are composed of both sparsely and heavily granulated cells that histochemically resemble serous acinar cells of the submandibular salivary gland. The cytoplasmic granules are strongly periodic acid-Schiff (PAS)-positive, and some are also stained by alcian blue for acidic mucosubstances. The lacrimal gland has a simple duct system of intralobular ducts and interlobular excretory ducts. Lymphocytes and plasma cells are common in the periductal stroma. Major lacrimal glands of rhesus monkeys are suitable for comparative and correlative studies of lacrimal and salivary diseases and radiation responses.     

Quantitation of immunoglobulin- and peptide hormone-producing cells in gastrointestinal mucosa. Comparison of direct immunofluorescence and the unlabelled antibody peroxidase--antiperoxidase method

Direct immunofluorescence (DIF) and the unlabelled antibody peroxidase--antiperoxidase (PAP) methods were compared on a quantitative basis with regard to visualization of IgA immunocytes and gastrin cells in human gastric mucosa, and secretin cells in canine duodenal mucosa. With both DIF and PAP, two serial sections from 13 biopsy specimens were evaluated for each cell type--thus keeping tissue preparation the same with both staining methods. The three cell types were well visualized regardless of method, and there was no significant difference between cell numbers recorded with the DIF or PAP. When blind duplicate counts were obtained with an interval of three weeks, comparisons of weighted differences and the Kendall's rank correlation test indicated good precision; the reproducibility of duplicate enumerations with each method was comparable to that between the two methods. It was concluded that DIF and PAP are equally applicable for studies of these three cell types under the conditions used in this investigation.     

Light microscopic triple-colored immunohistochemical staining on the same vibratome section using the avidin-biotin-peroxidase complex technique

The sequential application of the avidin-biotin-peroxidase complex technique was used to localize multiple tissue antigens on a single free floating section of rat brain. Sequential visualization of individual antigens was achieved by the silver-gold-intensified diaminobenzidine (DAB) in the first step, nickel-intensified DAB in the second step, and the DAB alone in the third step of the immunostain procedure. For the demonstration of this method, tyrosine hydroxylase (TH), corticotropin-releasing factor (CRF), and vasopressin (VAS) antisera were used. Sections from the hypothalamic paraventricular nucleus (PVN) of rats pretreated with colchicine were stained. Black TH containing cell bodies were clearly distinguished from blue stained CRF cells and from yellow stained VAS-containing cell bodies in the PVN on the 25-30 micron thick vibratome sections. The sequential immunostaining procedure presented here results in superior staining of multiple antigens as compared to that achieved by the sequential application of the peroxidase-antiperoxidase (PAP) technique.     

Glial fibrillary acidic protein and desmin in salivary neoplasms

The presence of intermediate filament proteins (IFP) in normal salivary gland tissue and in a number of salivary gland neoplasms has been investigated by immunohistochemical techniques on frozen sections. Cytokeratins (CKs) were seen in almost all normal epithelial cells. In the parotid gland and in palatal gland tissue, a co-expression of cytokeratin and glial fibrillary acidic protein (GFAP) was seen in some myoepithelial cells, but this was not apparent in the submandibular gland. In some pleomorphic adenomas, carcinomas in pleomorphic adenomas, one mucoepidermoid carcinoma, one mucus-producing adenopapillary carcinoma and one adenoid cystic carcinoma, cells expressing three different IFP classes were found (CKs, vimentin, GFAP). These cells were most often situated peripherally in the tumour cords or ducts. The cytokeratin pattern in these cells, as revealed by mAbs PKK1-3, was similar to that in normal myoepithelial cells. Furthermore, reactivity for a fourth class of IFP, desmin, could be seen in this cell type in two carcinomas in pleomorphic adenomas, and also in a few cells in a pleomorphic adenoma and an adenoid cystic carcinoma. Thus the pattern of IFP expression in salivary gland neoplasms, is very complex, and cannot always be related to the normal tissue.     

Magainin-like immunoreactivity in human submandibular and labial salivary glands

Magainins, antimicrobial peptides secreted by granular glands of frog skin, may be related to the high resistance to infections of this epithelial surface. The oral mucosa of healthy individuals is another tissue in which infection is not frequent, probably owing to the activity of potent salivary and mucosal defense mechanisms. To investigate if magainin-like factors are a component of these oral defense mechanisms, human and animal minor (mucosal) and major salivary glands were examined by immunohistochemistry, using a polyclonal rabbit anti-magainin antibody. Cryostat sections of (para) formaldehyde-fixed tissues were incubated with the antibody and then stained with fluorescein-complexed anti-rabbit IgG. Specific staining was observed in the apical portion of the cytoplasm of ductal epithelial cells of human submandibular and labial salivary glands. Diffuse staining was present in submandibular acinar cells. Bovine, rat, hamster, and mouse tissues were unreactive. The presence of magainin-like substances in human salivary gland duct cells is consistent with reports of the occurrence of other biologically active substances in salivary gland ducts.     

Distribution of Tight Junction Proteins in Adult Human Salivary Glands

Tight junctions (TJs) are an essential structure of fluid-secreting cells, such as those in salivary glands. Three major families of integral membrane proteins have been identified as components of the TJ: claudins, occludin, and junctional adhesion molecules (JAMs), plus the cytosolic protein zonula occludens (ZO). We have been working to develop an orally implantable artificial salivary gland that would be suitable for treating patients lacking salivary parenchymal tissue. To date, little is known about the distribution of TJ proteins in adult human salivary cells and thus what key molecular components might be desirable for the cellular component of an artificial salivary gland device. Therefore, the aim of this study was to determine the distribution of TJ proteins in human salivary glands. Salivary gland samples were obtained from 10 patients. Frozen and formalin-fixed paraffin-embedded sections were stained using IHC methods. Claudin-1 was expressed in ductal, endothelial, and ∼25% of serous cells. Claudins-2, -3, and -4 and JAM-A were expressed in both ductal and acinar cells, whereas claudin-5 was expressed only in endothelial cells. Occludin and ZO-1 were expressed in acinar, ductal, and endothelial cells. These results provide new information on TJ proteins in two major human salivary glands and should serve as a reference for future studies to assess the presence of appropriate TJ proteins in a tissue-engineered human salivary gland. (J Histochem Cytochem 56:1093–1098, 2008)     

Human salivary gland acinar cells spontaneously form three-dimensional structures and change the protein expression patterns

Applying tissue engineering principles to design an auto-secretory device is a potential solution for patients suffering loss of salivary gland function. However, the largest challenge in implementing this solution is the primary culture of human salivary gland cells, because the cells are highly differentiated and difficult to expand in vitro. This situation leads to the lack of reports on the in vitro cell biology and physiology of human salivary gland cells. This study used a low-calcium culture system to selectively cultivate human parotid gland acinar (PGAC) cells from tissues with high purity in cell composition. This condition enables PGAC cells to continuously proliferate and retain the phenotypes of epithelial acinar cells to express secreting products (α-amylase) and function-related proteins (aquaporin-3, aquaporin-5, and ZO-1). Notably, when the cells reached confluence, three-dimensional (3D) cell aggregates were observed in crowded regions. These self-formed cell spheres were termed post-confluence structures (PCSs). Unexpectedly, despite being cultured in the same media, cells in PCSs exhibited higher expression levels and different expression patterns of function-related proteins compared to the two-dimensional (2D) cells. Translocation of aquoporin-3 from cytosolic to alongside the cell boundaries, and of ZO-1 molecules to the boundary of the PCSs were also observed. These observations suggest that when PGAC cells cultured on the 2D substrate would form PCSs without the help of 3D scaffolds and retain certain differentiation and polarity. This phenomenon implies that it is possible to introduce 2D substrates instead of 3D scaffolds into artificial salivary gland tissue engineering.