Low temperature FESEM of the calcifying interface of a scleractinian coral

The ultrastructural nature of the calcifying interface in the scleractinian coral Galaxea fascicularis has been investigated using high-resolution, low temperature field emission scanning electron microscopy (FESEM). This technique permitted structural analyses of soft tissue and skeleton in G. fascicularis in a frozen-hydrated state, without the need for chemical fixation or decalcification. Structural comparisons are made between frozen-hydrated polyps and polyps that have undergone conventional fixation and decalcification. Vesicles expelled by the calicoblastic ectodermal cells into sub-skeletal spaces and previously suggested to play a role in calcification were commonly observed in fixed samples but were distinctly absent in frozen-hydrated preparations. We propose that these vesicles are fixation artefacts. Two distinct types of vesicles (380 and 70 nm in diameter, respectively), were predominant throughout the calicoblastic ectodermal cells of frozen-hydrated preparations, but these were never seen to be entering, or to be contained within, sub-skeletal spaces, nor did they contain any crystalline material. In frozen-hydrated preparations, membranous sheets were seen to surround and isolate portions of aboral mesogloea and to form junctional complexes with calicoblastic cells. The calicoblastic ectoderm was closely associated with the underlying skeleton, with sub-skeletal spaces significantly smaller (P<0.0001) in frozen-hydrated polyps compared to fixed polyps. A network of organic filaments (26 nm in diameter) extended from the apical membranes of calicoblastic cells into these small sub-skeletal cavities. A thin sheath was also frequently observed adjacent to the apical membrane of calicoblastic cells.     

Time Course of Cell Sheet Adhesion to Porcine Heart Tissue after Transplantation

Multilayered cell sheets have been produced from bone marrow-derived mesenchymal stem cells (MSCs) for investigating their adhesion properties onto native porcine heart tissue. Once MSCs reached confluence after a 7-day culture on a temperature-responsive culture dish, a MSCs monolayer spontaneously detached itself from the dish, when the culture temperature was reduced from 37 to 20°C. The basal extracellular matrix (ECM) proteins of the single cell sheet are preserved, because this technique requires no proteolytic enzymes for harvesting cell sheet, which become a basic building block for assembling a multilayer cell sheet. The thickness of multilayered cell sheets made from three MSC sheets was found to be approximately 60 μm. For investigating the adhesion properties of the basal and apical sides, the multilayered cell sheets were transplanted onto the surface of the heart’s left ventricle. Multilayered cell sheets were histological investigated at 15, 30, 45 and 60 minutes after transplantation by hematoxylin eosin (HE) and azan dyes to determine required time for the adhesion of the multilayered sheets following cell-sheet transplantation. The results showed that only the basal side of multilayered cell sheets significantly enhanced the sheets adhesion onto the surface of heart 30 minutes after transplantation. This study concluded that (1) cell sheets had to be transplanted with its basal side onto the surface of heart tissue and (2) at least 30 minutes were necessary for obtaining the histological adhesion of the sheets to the heart tissue. This study provided clinical evidence and parameters for the successful application of MSC sheets to the myocardium and allowed cell sheet technology to be adapted clinical cell-therapy for myocardial diseases.     

A Method for Embedding Thin Membranes in Historesin

A method is described for flat-embedding thin membranous tissues in Historesin. It allows easy orientation for sectioning large areas parallel to the surface. Selected fields can be monitored from the unfixed specimen, throughout preparation, to mounting on the microscope slide. For cross-sectioning, the flat-embedded tissue can be stacked and re-embedded to increase the amount of material examined per section.     

Using Scrapings from Formalin-Fixed Tissues to Diagnose Leptospirosis by Fluorescent-Antibody Techniques

Small specimens of formalin-fixed tissues approximatey 1 × 1 × 0.2 cm were cut from the suspect specimen. Several clean microscope slides were dipped in 1% aqueous gelatin and air-dried or dried on a slide warmer. Each tissue specimen was washed in running tap water for 2-5 min and then lightly scraped with a straight knife blade, cutting edge perpendicular to the surface of the specimen. The scrapings were allowed to build up and cling to the knife blade, which was then turned so that the broad surface contacted the slide; thus, the scrapings could be smeared onto the slide in a single motion. Sufficient pressure was applied to embed the tissue fragments in the gelatin coating. Smears, dried in air or on a slide warmer, were stained immediately by a standard direct or indirect technique to detect fluorescein-labeled antigens. This scraping method, adapted to the study of leptospirosis by fluorescent-antibody technique, could reduce the need for cryostat-cut tissues and facilitate the observation of individual leptospires.     

Aspects of the arrangement of the adrenergic innervation in guinea-pigs as revealed by the fluorescence histochemical method applied to stretched,air-dried preparations

Summary The suitability of stretched, air-dried preparations for the fluorescence histochemical localization of catecholamines has been examined for a range of organs, including atrium, arteries, veins, digestive tract, urinogenital organs and sympathetic ganglia of the guinea-pig. Where possible, the tissue is divided into thin sheets under a dissecting microscope. It is then stretched over a glass slide to which it adheres as it dries. Stretch preparations can be easily and quickly prepared for the fluorescence localization of catecholamines and give clear and consistent results with many adrenergically innervated tissues. This technique allows the branching of adrenergic nerve trunks and of individual fibres to be readily followed. The relationships between fluorescent cells and other tissue elements can be traced quickly in situations where serial sections through the tissue would otherwise be necessary.     

Vimentin antibodies stain membranous epithelial cells in the rabbit bronchus-associated lymphoid tissue (BALT)

Summary The lymphoepithelium covering the bronchus-associated lymphoid tissue (BALT) of the rabbit lung was studied with monoclonal antibodies against vimentin, using the indirect immunoperoxidase technique. In the lymphoepithelium single cells which had a membranous apical cytoplasm and engulfed intraepithelial lymphocytes were vimentin-immunoreactive. All other epithelial cells of the lymphoepithelium and of the surrounding airway epithelium did not bind vimentin antibodies. The results support the hypothesis that the membranous epithelial cells in the lymphoepithelium of rabbit BALT are analogous with intestinal M-cells, which in rabbit Peyer's patches and appendix are selectively labelled by vimentin antibodies.     

Vimentin antibodies stain membranous epithelial cells in the rabbit bronchus-associated lymphoid tissue (BALT).

The lymphoepithelium covering the bronchus-associated lymphoid tissue (BALT) of the rabbit lung was studied with monoclonal antibodies against vimentin, using the indirect immunoperoxidase technique. In the lymphoepithelium single cells which had a membranous apical cytoplasm and engulfed intraepithelial lymphocytes were vimentin-immunoreactive. All other epithelial cells of the lymphoepithelium and of the surrounding airway epithelium did not bind vimentin antibodies. The results support the hypothesis that the membranous epithelial cells in the lymphoepithelium of rabbit BALT are analogous with intestinal M-cells, which in rabbit Peyer's patches and appendix are selectively labelled by vimentin antibodies.     

STUDIES ON THE ORGANIZATION OF THE BRUSH BORDER IN INTESTINAL EPITHELIAL CELLS : II. Fine Structure of Fractions of Tris-Disrupted Hamster Brush Borders

Two of the fractions obtained by density gradient centrifugation of Tris-disrupted brush borders from hamster intestinal mucosa have been identified as the microvillus cores and their surrounding membranous coats, respectively. This identification has the following morphological basis. In shadowed preparations one fraction (cores) appears as rounded, compact rods, and the other fraction (coats) appears as flattened sheets. Both rods and sheets have dimensions appropriate to the identities assigned to them. In addition, negative staining shows that the rods are composed of aligned particles of roughly 60 A, consistent with the appearance of the core in tissue section, where 60-A fibrils are characteristic. The sheets are covered by non-aligned particles of approximately the same size. Sectioned preparations show that the core fraction contains predominantly fibrous material with some membranous contamination and that the coat fraction is apparently composed exclusively of elongated sacs with a unit membrane structure. Some details of the structure of the core are evident in cases where the compact rod appears to be loosened, revealing a doubled strand. The strand is approximately 350 A wide; the compact rod is roughly twice this width. With negative staining the strand shows a dense central region. The morphological identification presented here is consistent with the distribution of enzymic activity among the density gradient fractions described in the preceding paper.     

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section¹. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).     

Lamination of Thermoplastic Sheets as a Means of Mounting Histological Material

Permanent preparations of squashes, whole mounts and stained sections can be made by lamination of thermoplastic sheets. Classical procedures of staining and dehydration for sectioned material were used although dehydration after staining was not required for root tip squashes. Arranging the specimen with the identification label between two pieces of clear Vinylite plastic, 15 mils thick, tightening the preparation in a Photo-Seal Kit electric press and laminating for 3 min gave a finished preparation without the use of glass slides and cover slips. For root tip squashes, the stained tip was placed with a drop of stain between two pieces of plastic, squashed and then laminated. This insured retention of all the tissue which is sometimes lost during mounting processes. Preparations of unstained whole mounts were similarly laminated, with an identification label added between the plastic sheets. Stained sections were placed between two sheets of plastic but the identification label was placed on top of the preparation and a third piece of plastic added. This prevented the label from absorbing excess stain and the increased thickness allowed the slide to be used in a mechanical stage. Well preserved slides 18 mo old indicate that the laminated plastic slide is quite durable. It saves time, reveals good cytological detail and avoids some of the laborious features of other methods. It is a technic that can be used in introductory microtechnic and in the preparation of slides for class use in histology.     

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.     

Whole Mount Preparation of Chick Blastodermal Edge Tissue for Microscopic Examination

Three methods of preparation of chick blastodermal edge tissue for conventional microscopy were attempted: (1) Sections were cut from Water Wax (E. Gurr) embedded material. This method was unsatisfactory due to loss of morphological relationships produced by the inability to attach the tissue to a slide. (2) Frozen sections were cut from embryonic marginal tissue with its underlying white and yellow yolk which was embedded in a 25% gelatin solution. This method retained morphological relationships prior to 24 hr of egg incubation, but was technically impractical in excess of this period of incubation. Also, the gelatin could not be removed from the sections and cellular detail was obscured by its subsequent staining. (3) The blastoderm was removed from the yolk and adherent vitelline membrane, and the yolk dissected from a small piece of this blastodermal edge tissue under a dissecting microscope. The dissected tissue, primarily monocellular and dicellular in thickness, was transferred to a slide and attached to it by allowing a few drops of ether-alcohol (1:1) to flow over it. The plane of the tissue was, therefore, parallel with the plane of the slide. Most fixatives and staining techniques could subsequently be used. Fixing for 30-120 min in ether-alcohol (1:1) containing 0.5% glacial acetic acid gave excellent results with the staining techniques attempted. Fixation with Zenker's fluid for 24 hr followed by a 6 min hydrolysis with 1 N HC1 was best prior to the Feulgen technique.     

Ultrastructural organization of the epithelial layers and surface morphological characteristics of cells in adenocarcinomas of the cervix uteri

The cell interrelations, and cellular attachment to the stroma in normal columnar epithelium and adenocarcinoma of the cervix uteri were examined by transmission and scanning electron microscopy. The application of rapid enzymatic digestion technique allows to visualize the topography of cell membranes, otherwise disguised in ordinary conditions. Four types of disordered epithelial sheets characterized by different apical, lateral and basal cell surface changes are described. Various alterations in morphology of the basement membrane and adjacent conjunctive tissue are associated with the tumor appearance. Marked deviations in cell-stroma contact may lead to the inversion of cell polarity revealed in cervical adenocarcinoma: cellular parts adjoining to stroma acquire characteristic features of the apical pole.     

Spontaneous reversal of polarity of the voltage across LLC-PK1 renal epithelial cell sheets

While sterilely monitoring transepithelial voltage (potential difference) across LLC-PK cell sheets over a 24-hr period, we noted that the apical-negative, transepithelial voltage, a key property of the LLC-PK1 renal epithelial cell line, reverses polarity to become apical-positive. This spontaneous change of polarity of electrical potential difference (PD) across LLC-PK1 cell sheets cultured on permeable filters was observed to occur approximately 12 hr after refeeding. Unlike the apical (luminal)-negative PD, the apical-positive PD was insensitive to phlorizin and ouabain. Both were insensitive to the diuretics amiloride, furosemide, and 4-acetamido-4-isothiocynato-stilbene-2,2-disulfonic acid (SITS). A pH gradient existed across apical-positive cell sheets (apical medium more acidic by 0.3 units) but an osmotic gradient did not. Unlike the temperature-sensitive apical-negative PD, the apical positive PD was unaffected by brief exposure to 4 degrees C temperature. Junctional disruptive agents such as the tumor promotor, TPA, dissipated both types of PD with similar time courses. The formation of the apical-positive PD correlated in time with apical glucose levels falling below the reported Km of the Na+-sugar contransporter. A high glycolytic rate per se may not be essential for this PD polarity reversal since the reversal could occur in glucose-free medium with a normal time course and magnitude. The lysis with time of floating cells with consequent release of KCl into the apical compartment was also considered as a possible cause of the polarity reversal, but the turnover of even 2 X 10(6) cells in 12 hr was found not to raise apical KCl sufficiently to produce the polarity shift. Although a significant K+ gradient did not exist across cell sheets with apical-positive PD values, a sizable gradient of Cl- did exist, directed apical to basolateral. This gradient, coupled with anion-selective tight junctions, should contribute to the observed apical positive voltage. The voltage polarity shift seen in these cell cultures with time is not unlike the polarity shift occurring in the renal proximal convoluted tubule, with distance from the glomerulus.     

Graded Impregnation of Nervous Tissue Stained by the Golgi Procedure

A simple technique is described by which retinal whole mounts or slices of brain can be impregnated by the Golgi procedure in a graded manner. The unfixed tissue is laid on a glass slide and covered with a layer of Telfa gauze which is tied in place. Following fixation, the progress of staining, which begins beneath holes in the gauze, is followed by direct observation through the slide. The tissue is impregnated or reimpregnated until the desired results are obtained.     

Freezing by Liquid Carbon Dioxide in Making Slides Permanent

The expansion of liquid CO₂ may be employed in a quick-freeze method for making aqueous slide preparations permanent. An apparatus is described for this purpose which could be duplicated satisfactorily by cutting a 22mm square hole in the top of a standard freezing microtome specimen holder. The edges should be filed smooth to provide a flat surface for the slide to rest on, and clamps added to keep the slide in place while freezing. Once the slide is frozen, the cover slip may be readily removed, leaving practically all of the tissue on the slide. Following simultaneous thawing and dehydration of the slide in 95% alcohol, covering is done with Diaphane or Euparal and a clean, dry cover slip.     

Comparison between slide and flow cytophotometric DNA measurements in breast tumors

Nuclear DNA analysis was performed in 37 human mammary adenocarcinomas in order to elucidate the difficulties and pitfalls connected with the interpretation of DNA histograms obtained using different methodologic approaches. For each tumor, DNA profiles were obtained by means of slide microspectrophotometry on a fine needle aspirate, slide cytophotometry on a 4-micron histologic section and flow cytometry on a suspension prepared from a cube of fresh tissue. When the DNA histograms were interpreted according to criteria usually applied to discriminate low-grade malignant tumors from high-grade malignant tumors, some tumors classified as euploid by one method were classified as aneuploid by another method. The main reasons for this weak correlation seem to be in specimen preparation and in tumor cell representation within the specimen between the methods. Another reason is that slide and flow techniques exhibit different sensitivities for malignancy-associated nuclear DNA changes: minor alterations of the DNA content of the tumor stemlines seem to be more exactly reported by means of the flow technique whereas structural alterations of the nuclear chromatin seem to be more sensitively recorded by means of the slide technique. It is suggested that thorough control of each step of the various DNA analysis procedures and the use of information obtainable by slide and flow techniques taken together may significantly improve the prognostic value of DNA measurements.     

Preparation of Leaf Epidermis for Topographic Study

Epidermal strips of leaves of the Gramineae can be prepared using the following technique: The mature leaf is dipped in boiling water to kill the cells, and decolorized in boiling 70% alcohol. It is cleared and softened in 88% lactic acid. Epidermal, mesophyll and vascular tissue is removed from a selected constant area of the leaf leaving an epidermal strip 1-3 cm in length. This is inverted on a slide, stained in lactopheno-cotton blue, and destained in 88% lactic acid. Transverse and longitudinal sections of the strip are obtained at this stage. The epidermal strip is finally mounted on a slide in 88% lactic acid. The preparation is photographed with a 35 mm camera using transmitted light, and a yellow filter in the microscope lamp. Photomicrographs of known enlargement are then prepared from which accurate measurements can be recorded. The technique is applicable to both fresh and herbarium material.