Glycol Methacrylate Sections Research Articles

Carbohydrate cytochemistry of rhesus monkey tracheal submucosal glands

This study was designed to characterize the ultrastructure and carbohydrate content of secretory cells in submucosal glands of rhesus monkey and to compare this information with that available for humans. The tracheas from five adult monkeys were fixed by airway infusion, processed, and embedded for both light and transmission electron microscopy. Histochemical strains including alcian blue-periodic acid-Schiff, dialyzed iron, and high-iron diamine-alcian blue were applied to serial glycol methacrylate sections. The cytochemical stains used included periodic acid-thiocarbohydrazide-silver proteinate, high-iron diamine, and low-iron diamine. The glandular secretory cells were divided into four categories based on ultrastructure and location within the gland. Cells in the first category resembled the mucous cell of the surface epithelium and were located in ducts most proximal to the tracheal lumen. The second category consisted of cells that were located in distal ducts and contained large electron-lucent granules. The granules in both of these cell groups contained material that was periodate-reactive and sulfated. Cells of the third category contained granules that were either electron-lucent or electron-dense. These cells, which were difficult to characterize as either serous or mucous, were located in secretory tubules and acini and contained periodate-reactive glycoconjugates that were either sulfated or nonsulfated. The last category consisted mainly of cells that contained electron-dense granules that were lightly periodate-reactive or a few that were unreactive with any of the cytochemical methods used here.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcian blue method for attaching glycol methacrylate bone marrow sections to glass slides.

Detachment of glycol methacrylate sections from glass slides is a common problem during histochemical and immunohistochemical procedures, particularly when large or hard tissue sections are stained and when using caustic solutions, alcohols, or proteases.

An immunohistochemical characterization of rhesus monkey respiratory secretions using monoclonal antibodies.

A series of 12 monoclonal antibodies was made against mucous and serous components of rhesus monkey trachea. These antibodies were used to localize secretory products in the respiratory epithelium by immunohistochemical methods. Mucous cells of the tracheal surface epithelium and submucosal glands were shown by histochemical methods to be a homogeneous population containing periodate-reactive sulfated glycoconjugates. Immunohistochemical staining using the monoclonal antibodies on glycol methacrylate sections serial to those stained histochemically revealed a more antigenically heterogeneous population of secretory cells. Four distinct staining patterns were observed with the 12 monoclonal antibodies. Eight antibodies reacted with most, but not all, mucous and serous cells. Two antibodies recognized subpopulations of secretory cells. One antibody stained tracheal mucous cells with a concentration of reaction product on the luminal border, and one antibody stained only serous cells within the glands. Ten of the 12 antibodies were shown to react in an ELISA with the high molecular weight void volume containing mucous glycoproteins separated on a Sepharose CL-4B gel filtration column. None of the antibodies recognized blood group antigens. We conclude: (1) that production of a large panel of monoclonal antibodies to respiratory tract mucins from primates is feasible, (2) that the monoclonal antibodies will recognize epitopes on biochemically identifiable glycoproteins, and (3) that the intracellular mucous products of tracheal secretory cells exhibit greater heterogeneity than is detectable by conventional histochemistry.

Development of neuroepithelial bodies in intact and cultured lungs of fetal rats

Intact, 14- to 21-day fetal rat lung pairs, neonatal lungs, and cultured 15- and 16-day lung explants were examined in 2-micron-thick glycol methacrylate sections stained by PAS-lead hematoxylin. Selected stages were also studied in histochemical preparations for aliesterase and formaldehyde-induced monoamine fluorescence, as well as by scanning and transmission electron microscopy. Neuroepithelial bodies (NEBs) first appear in pseudoglandular lungs at 15 days in vivo as pyramidal groups of basal, diffusely lead-hematoxylin-positive cells in glycogen-depleted epithelium of main and lobar bronchi. By day 16, primitive NEBs occur within three to four generations of the terminal buds, and older, proximal bodies are larger and more distinctive than at 15 days. Aliesterase activity is first detected in basally located, developing NEBs on day 16. During the canalicular and alveolar sac periods, NEBs appear and mature on a proximal-to-distal gradient along the airway, as they do in developing rabbit and human lungs. As earlier-formed airways elongate, additional NEBs appear and supplement the population already present. By days 20-21, NEBs occur at all airway levels down to the bronchiolo-alveolar junctions, and many of the cells have discrete PAS- and lead-hematoxylin-positive, infranuclear granules. Near term some NEBs exhibit serotonin fluorescence after incubation in 5-hydroxytryptophan and have abundant, ca. 100-nm, electron-dense granules. These are concentrated toward the cell base like the stained granules visualized by light microscopy. Similar results were obtained from lungs placed in organ culture. From 2 days in culture to a time equivalent to term, NEB formation parallels that in vivo, indicating that developmental requirements are met in in vitro. Taken altogether, morphologic and cytochemical evidence suggests that NEBs of rats are functional in late fetal life and that their development is relatively independent of extrapulmonary influences and of the intraepithelial ingrowth of sensory nerve endings.

Comparative evaluation of diagnostic cytological features of malignancy in GMA sections, paraffin sections and smears

A quantitative evaluation of glycol methacrylate (GMA) embedded sections in the cytological assessment of body fluids is reported for the first time. The technique is compared with routine smears, filter preparations and paraffin sections of cell blocks. Diagnostic problems were encountered mainly in the assessment of reactive mesothelial cells and lymphoreticular cells. Diagnostic accuracy was highly dependent on technique of cell preparation, and the GMA sections were a useful supplementary technique in cases where there were difficulties of interpretation. The method offers the advantage of improved sampling by using a concentrated cell block technique and high resolution of cellular detail essential for accurate cytological diagnosis.

A comparative study of softeners and catalyst systems upon dimensional changes and sectioning quality of glycol methacrylate sections

SUMMARYIt is shown that softeners and temperature have an important influence upon the stretching parameters of glycol methacrylate sections. Also it is demonstrated that the catalyst system used may significantly interfere with section stretching.

Glycol Methacrylate for Histology: Problems and Solutions

Four years of experience with plastic-embedded tissue sections are reported. A procedure for eliminating heat artifact, some modifications of staining procedures, and a consistently reliable and reproducible connective tissue trichrome stain for glycol methacrylate sections are described.

Restoration of mucociliary tracheal epithelium following deprivation of vitamin A. A quantitative morphologic study.

In order to learn more about the respective roles played by basal cells and mucous cells in the maintenance of tracheal mucociliary epithelium, cell kinetics and epithelial cell morphology were characterized over a 7-day period, during which dietary vitamin A was restored to previously deprived hamsters. Hamsters were reared from birth to 35 days of age on vitamin A-replete or deficient diets. Deprived hamsters were made replete by 5 mg vitamin A-acetate orally, plus a vitamin A-replete diet. Colchicine and 3HTdR were given 6 h before death. The numbers of basal cells, mucous cells, preciliated cells and ciliated cells, and mitotic rates (MR) and labeling indices (LI) of basal cells and mucous cells, were quantified in glycol methacrylate sections stained with PAS-lead hematoxylin. Vitamin A-deprivation decreased replication of basal cells and mucous cells in tracheal epithelium which showed minimal morphological change. The proportion of basal cells was increased and proportions of mucous, preciliated and ciliated cells were decreased. Following restoration of vitamin A to the diet, the basal cell MR remained below control level throughout the experimental period, but the mucous cell MR started to rise on day 2-replete, and on day 3-replete and thereafter the mucous cell MR was within the control range. Basal cell and mucous cell LI's showed similar trends. Preciliated cells were reduced or absent in vitamin A-deprived epithelium. Their number had risen by day 3-replete and thereafter they were generated within the control range. These cells matured into ciliated cells. By day 4-replete, the proportion of basal cells had decreased markedly and the proportions of mucous cells, and preciliated plus ciliated cells had increased, so that at this time cellular proportions were within or near control values. This trend continued so that by day 7-replete, a nearly normal mucociliary epithelium was restored. The results show that vitamin A-levels modulate replication rates of basal cells and mucous cells and indicate that mitotic division of mucous cells is a prerequisite for the genesis of preciliated cells and new mucous cells and for restoration of the mucociliary epithelium following deprivation of vitamin A in the diet.

Thickness variations within individual paraffin and glycol methacrylate sections.

The object of this study was to investigate whether there are intra-section thickness variations in individual paraffin and glycol methacrylate (GMA) sections. Using steel or glass knives sections were cut from liver and urinary bladder. Section thickness variations were measured with an interference microscope and amounted to 1-3 microns within individual paraffin sections and 0.3 microns within GMA sections. The results were confirmed by observations on sections which had been re-embedded and re-sectioned. Some of the variations within the paraffin sections were associated with the cell nuclei. It is concluded that GMA sections are much smoother than paraffin sections and thus more suitable for quantitative histological studies.

Further Use of the SMB (Silver Methenamine-Borate) Impregnation: Pneumocystis Carinii

"Further Use of the SMB (Silver Methenamine-Borate) Impregnation: Pneumocystis Carinii." Journal of Histotechnology, 6(2), p. 70

Changes in secretory cells of hamster tracheal epithelium in response to acute sublethal injury: a quantitative study.

Secretory cells are reported to rapidly increase in number when the tracheobronchial epithelium is irritated. We suggest that this acute change results from accumulation of secretion granules within specialized albeit unobstrusive secretory cells rather than from conversion of undifferentiated cells to secretory cells of the production of new cells by mitosis. This hypothesis was tested in hamster tracheal epithelium 6, 12, and 24 hr after intratracheal instillation of elastase in saline or saline alone. Basal, secretory, and ciliated cells and cells of indeterminate character were quantified by counting 1400 cells per hamster in 2 micrometers thick glycol methacrylate sections stained with periodic acid-Schiff (PAS)-lead hematoxylin. Secretory cells were characterized and quantified depending upon distribution patterns of the PAS-positive secretion granules and on the amount of intracellular secretion product. Thirty-two hamsters were studied; half of them received colchicine 6 hr prior to tissue sampling. Over 24 hr the percentage of ciliated cells showed no significant change and no morphological evidence of alteration was observed in this cell population; thus ciliated cells were excluded from the analysis. When the ciliated cell population was excluded, control and experimental values for the different cell categories remained statistically constant, if unavoidable classification errors at the light microscopic level were compensated for on the basis of ultrastructural features. Average control values for the non-ciliated cell population were: mitotic index 0.030, basal cells 27.8%, secretory cells 57.9%, and cells of indeterminate character 14.3%. Secretory product was decreased in secretory cells at 6 hr but thereafter secretion accumulated in these cells. At 24 hr the overall number of secretory cells appeared to be increased. However, the increase was apparent and not real and was accounted for by accumulation of secretion granules in preexisting but previously unobtrusive secretory cells.

Recovery from ozone-induced injury in the lungs of the Syrian golden hamster

In order to examine the morphological and cytokinetic changes in the hamster pulmonary epithelium after a high dose of ozone, Syrian golden hamsters were exposed to 15 ppm of ozone for 6 hr and killed 2 hr to 96 days later. Two hours before death, each animal was injected intraperitoneally with tritiated thymidine. Autoradiographs were prepared from 1-μm glycol methacrylate sections of lung tissue. Ozone produced immediate epithelial desquamation in all airways, leaving a single layer of either squamous cells or nonciliated cells which began to proliferate within 1 day of the exposure. The overall morphology of the airway epithelium returned to normal within 7 days. Damage in the alveolar region was localized to alveoli near the terminal bronchioles and consisted of desquamation of Type I epithelial cells and endothelial cells and the presence of inflammation. Hyperplastic regions of cuboidal, squamous, and ciliated cells formed at the bronchiolar-alveolar junction within 2 days of exposure. The greatest increase in proliferation was seen in airway epithelial cells which reached a maximal labeling index of 50%. We conclude that the hamster pulmonary epithelium has a great proliferative capacity to repair damage due to a high concentration of ozone. Foci of hyperplasia in both airway and alveolar epithelium appear during this repair process.

Comparison of Glycol Methacrylate-and Paraffin-Embedded Tissues for identification of Tritium-Labeled Cells after Emulsion Radioautography

High resolution radioautographs were obtained from tissue sections embedded in glycol methacrylate (GMA). These sections were of superior quality compared to paraffin-embedded sections and they greatly enhanced the precision by which the number and type of tritium-labeled cells in tissues could be identified. Mice were injected with 3H-thymidine (TdR) (0.5 μCi/gram body wt., 60 Ci/mMole) intraperitoneally and killed one hour later. Samples of spleen, testis, small intestine and distal colon were removed, washed, fixed in 1 % glutaraldehyde-4% formaldehyde, dehydrated, and embedded in GMA. Glycol methacrylate-embedded tissues were sectioned at 2 μm on a JB-4 microtome and floated onto glass slides. Comparable samples of tissue were embedded in paraffin and sectioned at 5 μm. Those sections that were to be used for routine analysis of the number or position of 3H-TdR-labeled cells were prestained by the Feulgen reaction, dipped in Kodak NTB-3 emulsion, exposed for 21 days at 4°C and developed. For high resolution radioautography in which both nuclear and cytoplasmic detail were desired, the sections were cut at 1 to 2 μm, prestained with Feulgen and then with 1 % alcian blue 8 G S in 3% glacial acetic acid for mucopolysaccharides and radioautographed. Cytoplasmic detail was further enhanced by poststaining the radioautographs in Lee's methylene blue-basic fuschin.The results indicated that (1) 2 μm GMA sections were easily obtained, (2) routine pre- and poststaining were achieved without fading or chernographic effect, and (3) radioautographs of tissue sections revealed that the cells were not distorted and labeled and unlabeled cells could be clearly distinguished. This technique is a marked improvement over paraffin-embedded sections for the production of high resolution radioautographs.

An unusual bronchial carcinoid tumor analyzed by conjunctive staining

Two staining methods selective for pulmonary APUD endocrine cells-PAS-lead hematoxylin and a more elaborate procedure termed conjunctive staining-were applied to 2 μm. glycol methacrylate sections of a human bronchial carcinoid tumor, in order to display the advantage they hold over routine pathologic methods in revealing the presence as well as cytochemical characteristics of endocrine cell granules in the tumor cells. PAS-lead hematoxylin alone showed the tumor population to be mixed, with a wide range of staining in the granules, extending between the extremes of PAS only and lead hematoxylin only. The conjunctive staining protocol is described in detail. In tissue previously reacted for argyrophilia, it demonstrates, sequentially, fluorescence for serotonin, PAS alone, and PAS-lead hematoxylin, all in a single section. By comparing photomicrographs of a given field recorded during the process, exact point by point correlations of staining behavior can be made, limited only by the resolving power of the microscope. Conjunctive staining of the tumor revealed 10 distinct cytochemical cell signatures, far exceeding the number of types of endocrine cells known to exist in normal human lung. Preliminary results indicate that three types with PAS and lead hematoxylin positive granules may occur in adult human bronchi, although previous studies have reported but a single type of endocrine cell. Numerous tumorlets in the bronchial epithelium overlying the tumor were an unusual feature of the case. These were composed of cells staining similarly to cells in the tumor mass. Some were connected to it by cords of cells, whereas other tumorlets were interconnected in the epithelium, but a number of smaller ones were clearly isolated from other tumor cells, as shown by serial sections. A few tumorlets contained cells with both mucous and lead hematoxylin positive granules. With a population density that exceeded the expected distribution of normal small granule endocrine cells in the bronchial epithelium and including cells with mixed endocrine and nonendocrine characteristics, the tumorlets provided testimony favorable to the view that carcinoid tumors like this one arise from indifferent cells of the bronchial epithelium that have been driven by oncogenic forces toward expressing an APUD cell phenotype.

A Stain for the Simultaneous Demonstration of Beta Cell Granules and Amyloid in Glycol-Methacrylate-Embedded Biopsy Samples of Pancreas fromMacaca nigra

Amyloid and beta cell granules were demonstrated simultaneously in glycol methacrylate sections of pancreatic tissue. The method involved the use of the aldehyde fuchsin technique of Mowry to color beta cell granules purple, and the use of the Alcian blue technique of Lendrum to color amyloid turquoise. Solution preparations and staining procedures were easily adapted from existing paraffin techniques.

Light and electron microscopical combined staining by immunohistochemical and enzyme histochemical methods using rat prolactin-secreting pituitary tumours.

Combined immunohistochemical staining (IHCS) and enzyme histochemical staining (EHCS) methods for light microscopy (LM) and electron microscopy (EM) are reported, using oestrogen-induced rat pituitary tumours. For LM, combined staining for alkaline phosphatase and acid phosphatase by EHCS, using the azo dye method, and for prolactin and ACTH by IHCS, using the enzyme-labelled antibody method, gave the best results on 1 microgram glycol methacrylate sections. For EM, combined staining by EHCS on 30 microgram tissue sections followed by IHCS for prolactin on ultrathin Epon sections (enzyme-labelled antibody method) provided acceptable results. By these combined staining methods, the neoplastic prolactin cells were shown to have close affinity to rich alkaline phosphatase-positive capillaries and to possess an alkaline phosphatase-positive cell membrane. Furthermore, they revealed acid phosphatase-positive lysosomal and secretory granules. These combined staining methods may be valuable in studies on the actual functional status of cells.

Ultrastructural visualization of concanavalin A binding sites using alkaline phosphatase-labeled Con A and ultrathin glycol methacrylate sections.

A method for the visualization of concanavalin A (Con A) binding sites by electron microscopy of glycol methacrylate sections is presented. This method, which is an application of the alkaline phosphatase-labeled Con A conjugate technique, solves the problems not only of limited penetration of chemicals into tissue blocks but also of injuries to tissue sections due to irritative reagents experienced in Con A-peroxidase staining. Glycol methacrylate sections are incubated successively with an alkaline phosphatase-labeled Con A solution and a lead citrate medium for the enzyme activity. Different kinds of tissues from adult rats have been used to test the method; tracheal cartilage, aorta and jejunum. The localization of Con A binding sites demonstrated by this method is consistent with the results of other published studies. Appropriate controls have been performed (ie., omission of the conjugated lectin, lectin plus its inhibitor) and these substantiate the specificity of the method.

The gingival epithelium of rodent molars with limited eruption.

We have re-examined the arrangement of epithelia surrounding molar teeth with limited eruption in the rat, mouse and hamster. Our methods permitted enamel to be retained in paraffin and glycolmethacrylate sections, so providing optimal preservation of epithelial relationships. Three basic patterns of epithelial arrangement were observed on the various aspects of molars. Non-keratinized junctional epithelium covers the interdental septum and the base of all gingival crevices even in areas of gingival recession. In all areas other than the interdental septum, a fold of keratinized gingival epithelium lies beneath the lateral aspects of junctional epithelium. The buccal and lingual aspects of interdental septa show a zone of transition between these two basic types of epithelial arrangement. In intact gingiva the junctional epithelium can be seen to extend a considerable distance on to enamel surfaces, with the result that actual gingival crevice depths are even less than previously assumed. Artefacts resulting from loss of enamel during processing are discussed in the light of previous attempts to explain epithelial arrangements in rodent gingivae.

Hematoxylin toluidine blue-phloxinate staining of glycol methacrylate sections of retina and other tissues.

For a detailed study of the developing chick retina a technique has been developed using glycol methacrylate embedding and a hematoxylin toluidine blue-phloxinate stain. After removal of the vitreous body, one half-segment of the eye is dehydrated through graded ethyl alcohols to 95%, infiltrated and embedded in glycol methacrylate, and sectioned at 2 micrometer. The sections are stained in alum hematoxylin and then in a mixture containing toluidine blue-phloxinate from a stock solution of the dye that has matured for 2-3 weeks. Differentiation is not required and there is only slight staining of the plastic matrix. The quality and clarity of the sections contrasts markedly with that of similarly stained 5 micrometer paraffin wax sections of the retina. This technique has also been applied to skin, spinal cord, dorsal root ganglia, pancreas and small intestine. The stained sections from these tissues have proved very useful in revealing structural components.

Glycol methacrylate embedding and staining

Abstract Histopathology and research laboratories, searching for an embedding medium that yields light microscopic resolution not before available with traditional paraffin methods have turned to plastics. Glycol methacrylate (GMA) embedding has proved to be useful in helping to solve the problem of greater resolution and has the advantage of less tissue shrinkage as well as speed and convenience in handling. The embedding medium is not removed before staining and most staining procedures are easily adapted to the GMA sections.