Improved cytologic localisation ofβ-glucuronidase in sections treated with ethanol

Summary Treatment of fixed frozen sections of rat kidney with 40–60% ethanol for 2 minutes at 0° C improved the cytologic localisation of-glucuronidase in naphthol AS-BI post-coupling technique. Concentration of ethanol, duration of treatment and temperature are critical. Higher concentrations of ethanol, longer treatment and higher temperatures deteriorate the normal localisation of the enzyme. Such pretreatment can he introduced as a step in the original histochemical technique.     

Identification of muscle fiber types in “semithin” sections stained with p-phenylene-diamine

Summary Semithin sections of osmium-fixed plastic embedded muscle, stained with p-phenylene-diamine, show three types of muscle fibers. Electron microscopy of adjacent sections indicates that differing content and distribution of mitochondria and fat vacuoles are the basis for this distinction.     

Localization of κ-casein on thin sections of casein micelles by the gold method

Summary The ultrastructural location of -casein in bovine casein micelles was investigated by the protein A-gold method. Casein micelles, fixed in glutaraldehyde, were embedded at low temperature to enhance immunocytochemical marking of thin sections. -Casein was found distributed throughout the micelles of all sizes with a higher concentration in the smaller micelles. No peripheral location of -casein was observed, even in the larger micelles. These results do not agree with coat-core structures proposed for casein micelles. However they favor models where -casein is distributed uniformly throughout the micelles.     

Localization of glycosylated κ-casein on thin sections of casein micelles by lectin-labelled gold markers

Summary The location of the glycosylated part of κ-casein in bovine casein micelles was investigated using gold particles (6 nm in diameter) labelled withRicinus communis lectin andLimulus polyphemus lectin. The pattern of marking of thin sections of micelles was similar with both lectins. Glycosylated κ-casein was distributed uniformly throughout most micelles of all sizes. Peripheral location of glycosylated κ-casein was observed only occasionally in some of the largest micelles. Quantitative data indicated that the concentration of the glycosylated protein was constant in micelles of increasing sizes. As larger micelles contain less total κ-casein than smaller ones, these data indicated that a greater proportion of their κ-casein is glycosylated. These results support models for casein micelle structure where κ-casein is distributed throughout the micelles. They do not agree with “coat-core” structures.     

Immunocytochemical detection of peroxisomal β-oxidation enzymes in cryostat and paraffin sections of human post mortem liver

Summary The immunocytochemical visualization of the peroxisomal -oxidation enzymes was investigated in three human post mortem liver samples. Acyl-CoA oxidase, bifunctional protein and 3-oxoacyl-CoA thiolase remained immunocytochemically detectable 30, 55 and 72 h after death. Peroxisomes in the parenchymal cells were clearly visualized for light microscopy (paraffin and cryostat sections), using protein A-gold in combination with silver enhancement. In two samples catalase activity became very weak, but catalase antigenicity was well preserved. The findings prove the diagnOstic value of post mortem samples, even after extreme conditions of tissue conservation. The technique of immunocytochemical staining for the peroxisomal -oxidation enzymes on unmounted cryostat sections has not been reported previously. This method allows a quick diagnOsis of biopsies from patients suspected of peroxisomal disorders.     

Lattice images from ultrathin sections of cellulose microfibrils in the cell wall of Valonia macrophysa Kütz.

The crystalline ultrastructure and orientation of cellulose microfibrils in the cell wall of Valonia macrophysa were investigated by means of high-resolution electron microscopy of ultrathin (approx. 28 nm) sections. With careful selection of imaging conditions, ultrastructural aspects of the cell wall that had remained unresolved in previous studies were worked out by direct imaging of crystal lattice of cellulose microfibrils. It was confirmed that each microfibril is a single crystal having a lateral dimension of 20·20 nm², because lattice images of 0.39 nm resolution were clearly recorded with no major disruption in the whole area of the cross section of the microfibril. There was no evidence for the existence of 3.5-nm elementary fibrils which have been considered to be basic crystallographic and morphological units of cellulose in general. It was also confirmed that the axial directions (crystallographic fiber direction) of adjacent microfibrils in each single lamella of the cell wall are opposite to each other.     

Demonstration of 5′-nucleotidase activity in unfixed cryostat sections of rat liver using a combined light- and electron-microscope procedure

Summary In the present study a technique was developed to demonstrate 5′-nucleotidase activity in unfixed cryostat sections of rat liver at the light- and electron-microscope level using a semipermeable membrane. In order to retain the ultrastructure of the unfixed material as much as possible, incubations were also performed at 4°C rather than at 37°C. The optimized incubation medium contained 300 mm Tris-maleate buffer, pH 7.2, 5 mm adenosine monophosphate as substrate, 30 mm cerium chloride as capturing agent for liberated phosphate, 10 mm magnesium chloride as activator and 1.5% agar. At the light-microscope level, similar localizations of 5′-nucleotidase activity were obtained when incubations were performed at 37°C and 4°C. Enzyme activity was present mainly at bile canalicular membranes and at sinusoidal membranes of hepatocytes; total activity was higher in pericentral than in periportal areas. Cytophotometric analyses revealed that specific formation of final reaction product (FRP) (test minus control reaction) at 37°C followed a hyperbolic curve with time. A linear relationship was found between specific amounts of FRP and section thickness up to 8μm. 5′-Nucleotidase activity was about three-fold higher after incubation for 30 min at 37°C than at 4°C. At the electron-microscope level, it was demonstrated that the ultrastructure of rat liver was rather well-preserved after incubating unfixed cryostat sections attached to a semipermeable membrane and electron-dense FRP was found at bile canalicular and sinusoidal plasma membrane of hepatocytes. The most distinct changes in ultrastructure after incubation at 37°C, in comparison with that at 4°C, were the appearance of multi-lamellar structures at bile canaliculi at 37°C. We conclude that the present method is valid for the demonstration of 5′-nucleotidase activity in unfixed cryostat sections of rat liver at both the light- and electron-microscope levels and that hypothermic incubations improve ultrastructural morphology substantially.     

A method for preparing 2- to 50-µm-thick fresh-frozen sections of large samples and undecalcified hard tissues

This article describes a method for preparing 2- to 50-micron-thick fresh-frozen sections from large samples and completely calcified tissue samples. In order to perform the more routine work involved, a tungsten carbide disposable blade was installed to a heavy-duty sledge cryomicrotome. An entire 10-day-old rat and bone and tooth samples from a 7-month-old rat were rapidly frozen. The frozen samples were attached to the cryomicrotome stage. The cutting surface of the samples was covered with a polyvinylidene chloride film coated with synthetic rubber cement and cut at -25 degrees C. The soft tissues and the hard tissues were satisfactorily preserved and all tissue cells were easily identifiable. Enzymatic activity in the fresh sections was much stronger than that in chemically fixed and/or decalcified sections. The sections permitted histological and histochemical studies without trouble. In addition, the sections can be used for multiple experiments such as immunohistochemistry, in situ hybridization, and electron microprobe X-ray micro-analysis. This method can be used with conventional cryomicrotome equipment.     

A quantitative cytochemical method for the demonstration of Β5,3β-hydroxysteroid dehydrogenase activity in unfixed tissue sections of rat ovary

Summary A method for the quantitative measurement of ⁵, 3-hydroxysteroid dehydrogenase activity in unfixed tissue sections of rat ovary has been described. The method depends on the oxidation of dehydroepiandrosterone (DHEA) and uses nitroblue tetrazolium as the final electron acceptor. Although the dehydrogenase is not a soluble enzyme, polyvinyl alcohol is included in the reaction medium to allow the use of a high substrate concentration whilst employing a low concentration (5%) of dimethyl formamide. The enzyme is equally dependent on NAD⁺ or NADP⁺ for its activity and this activity is significantly enhanced by the presence of cyanide. The NADP⁺ dependence is not abolished by inhibiting nonspecific alkaline phosphomonoesterase. The activity of ⁵, 3-hydroxysteroid dehydrogenase is completely dependent on a functional sulphydryl group. Furthermore, the enzyme activity is totally inhibited in the presence of a steroid substrate analogue at 10^–4 M.     

Alkali-catalyzed β-elimination of periodate-oxidized glycans: A novel method of chemical deglycosylation of mucin gene products in paraffin embedded sections

Altered expression of mucin gene products has been described in many epithelial cancers including colorectal cancer. However, mucins are heavily O-glycosylated making the study of apomucin expression difficult. In this study, we describe a novel method of chemical deglycosylation of mucin gene products on paraffin embedded formalin-fixed tissue sections. In the normal and cancerous colorectum, our results suggest that alkali-catalyzed -elimination of periodate oxidized glycan method of chemical deglycosylation modifies the structure of carbohydrates sensitive to mild periodate oxidation resulting in less steric hindrance and selectively removes Tn and sialyl-Tn structures, partially exposing the underlying apomucin epitopes. Using this method, we have demonstrated that the MUC1 tandem repeat epitope recognized by MAb 139H2 is masked predominantly due to steric hindrance by carbohydrate structures whereas the MUC2 tandem repeat epitope recognized by MAb CCP58 and pAb MRP and the MUC3 tandem repeat epitope recognized by pAb M3P are masked by the presence of carbohydrate side chains O-linked to Ser/Thr residues within the epitope. Considerable differences in the level and pattern of expression of the epitopes in the tandem repeat region of apomucins of MUC1, MUC2, and MUC3 were observed between normal and cancerous colorectal cancer tissues. We conclude that this novel chemical deglycosylation method that causes selective cleavage of distinct glycans will be useful in unmasking various mucin gene products and glycoproteins containing similar O-glycosidic linkages in the tissue sections of formalin-fixed paraffin embedded normal and pathological tissues.     

One antigen, one gold? A quantitative analysis of immunogold labeling of plasma membrane 5'-nucleotidase in frozen thin sections

We present an evaluation of the efficiency of immunogold labeling for a low abundance plasma membrane protein. Several independent methods were used to determine the density of 5'-nucleotidase on the plasma membrane of the Fao cell. These methods include morphometry in combination with either enzymology or cell surface radiometric assay. Immunocytochemistry of frozen thin sections with either single or double layers of antibody and visualized with protein A complexed with 5 nm colloidal gold was used to estimate the same density. The application of a balance sheet to immunogold labeling demonstrates that the labeling is never quantitative. For example, labeling of the cell surface is always greater than labeling on the section. We show that departures from the "one antigen, one gold" ideal are systematic, so that an efficiency can be calculated and quantitative results can be obtained. The ability to obtain reliable quantitative results from immunogold labeling extends the utility of this already powerful technique.     

An improved method to detect β- galactosidase activity in transgenic mice: a post-staining procedure on paraffin embedded tissue sections

The Escherichia coli -galactosidase gene is frequently used as a reporter gene in transgenic studies because its activity can be easily detected at the cellular level. Here we report a procedure for monitoring -galactosidase activity directly in tissue sections, which involves the use of a mixture of ethanol and poly-ethylene-glycol as a fixative (Kryofix) and a special paraffin characterized by a lower fusion point of 42 °C. After embedding and cutting, the sections are stained by the chromogenic substrate 5-bromo-4-chloro-3-indoyl--d galactopyranoside (X-Gal). This procedure allows both the retention of a high level of -galactosidase activity and the preservation of good tissue morphology. Furthermore, it can be combined with immunohistochemical methods to detect other cellular components without compromising reporter gene detection     

The metabolism of N6(Δ2-isosopentenyl) [3H]adenine by different stem sections of Pisum sativum

Serial segments of internodal stem tissue were isolated from Pisum sativum L. shoots and incubated in a medium containing N⁶(²-isosopentenyl) [³H]adenine. The recovery of radioactive derivatives separated using HPLC indicated a gradient of cytokinin metabolic activity in the stem. This gradient of activity was found to be greatest at the basal node in young seedlings but was high both at upper and lower nodes in older plants. An attempt to correlate this phenomenon with the basipetally decreasing concentration of indole-3-acetic acid in the stem led to an experiment in which stem segments were pretreated in indole-3-acetic acid solutions before incubating in a medium containing N⁶(²-isosopentenyl) [³H]adenine. Indole-3-acetic acid was found to have a marked effect on cytokinin metabolism in isolated stem segments. These results are discussed in relation to apical dominance in the shoot.     

Facies and diagenetic evaluation of the Permian–Triassic boundary interval and basal Triassic carbonates: shallow and deep ramp sections,Hungary

The Permian–Triassic boundary and basal Triassic shallow-marine successions were studied and correlated in sections of two structural units in Hungary (Transdanubian Range and Bükk units). Core sections in the Transdanubian Range unit recovered inner ramp deposits whereas outcrops in the Bükk unit expose deposits of the deeper ramp area of the western Tethys. The inner ramp section (studied ca. 10 m in thickness) is characterized by a succession of dolomites overlain by bioclastic limestones, peloidal grainstones (which recorded the biotic decline) and oolites with finely crystalline limestone interlayers. The deeper ramp section (studied ca. 15 m in thickness) is characterized by a succession of bioclastic limestones and marlstones, mudstone beds (recording the first biotic decline), the ‘boundary shales’ (recording the second biotic decline and the stable carbon isotope marker), mudstones with wackestone laminae, and stromatolite boundstones. Accordingly, oolite formation and microbial micrite precipitation represent carbonate sedimentary responses of end-Permian mass extinction on the carbonate shelf. In both successions, mudstones predominate the upsection, suggesting a relative sea-level rise. The succession of the deep ramp area exhibits a continuous sediment accumulation and the diagenesis here was influenced by marine and marine-derived pore water. The δ¹³C curve shows a continuous change towards more negative values, starting in bioclastic limestones, followed by a sharp symmetric negative peak at the second biotic decline that is a chemostratigraphic marker of the boundary event. Facies and microfacies trend of the inner ramp carbonates in the Transdanubian Range unit exhibits close similarities to that found in many South Alpine sections. Relict peloidal deposits, formed cemented submarine hardground substrate, indicate the extinction level. Sedimentary and diagenetic features of the overlying oolite bedset revealed slightly different depositional environments in the two studied Transdanubian Range unit sections. Petrography of the oolites highlighted shallow burial diagenetic alterations which includes marine cementation, marine-burial replacement and dolomitization. A lack of the specific negative peak in the δ¹³C values is most likely due to the multiple redeposition events of the sedimentary grains. This led to the conclusion that the deeper ramp deposits (e.g., in Bükk unit) have greater potential for recognizing trends in processes, affecting the marine environments and related to the end-Permian mass extinction, at the western Tethys.     

Basic and 'special' stains for plastic sections in bone marrow histopathology, with special reference to May-Grünwald Giemsa and enzyme histochemistry

A battery of morphological, histochemical, and enzyme histochemical stains have been experimented on semithin sections of glycol-methacrylate-embedded bone marrow biopsies. We have been able to reproduce on sections the typical 'Romanowsky effect' which characterizes May-Grünwald Giemsa-stained smears of bone marrow or peripheral blood. This appears to be of critical importance for proper routine morphological evaluation of bone marrow biopsies. Conventional histochemical stains, and the enzyme histochemistry reactions that are most useful and widely used in the study of marrow aspiration smears have been successfully applied to plastic sections: in this way the evaluation of the cytochemical profiles of marrow diseases, especially leukemias, may be included in the histopathologist's diagnostic approach, with the additional advantage of preserving the architecture of the tissue and the relationship between haemapoietic cells and stromal components.     

Comparative FISH analysis of numerical chromosome 7 abnormalities in 5-μm and 15-μm paraffin-embedded tissue sections from prostatic carcinoma

 Interphase fluorescence in situ hybridization (FISH) was performed on 15-μm-thick paraffin sections from prostatic carcinomas using a chromosome 7-specific α-satellite DNA probe. A confocal laser scanning microscope (CLSM) was used for optical sectioning of the thick sections and reconstruction of 3D images. The number of FISH signals was determined by a gallery of optical sections evaluating only complete nuclei. To investiate the influence of section thickness and truncation and nuclei on scoring results, we compared the FISH data from 15-μm sections with signal counts obtained from 5-μm sections. The latter were evaluated by conventional fluorescence microscopy in the same tumor regions previously defined and marked on the slides. After statistical analysis of spot frequencies in tumor and non-tumorous cells (χ² test), we transferred the signal frequencies into a cytogenetic classification (−7, +7, polysomy 7). Based on this classification, most cases showed more than one chromosome 7 aberration type. Trisomy 7 (+7) became apparent in 15-μm-thick sections in all 19 tumors, polysomy 7 (>3 spots) in 18/19 cases, and monosomy 7 (−7) in 13/19 cases. In 5-μm sections, however, trisomy 7 and polysomy 7 were found in only 7/19 and 13/19 cases, respectively, and monosomy 7 in 7/19 cases. When comparing the classification results of tumor cells of the same tumor regions originating either from 5-μm or 15-μm sections, the following discrepancies were noted: in 15-μm sections exclusively, in 12/19 tumors, trisomy 7 was found; in 6/19 cases, polysomy 7; in 8/19 cases, monosomy 7. The high proportion of cases with tumor nuclei expressing only one hybridization signal of chromosome 7 in 15-μm sections could be confirmed as monosomy 7 in five selected cases by double-hybridization using centromere-specific probes for chromosomes 7 and 12. These results demonstrate that numerical chromosome 7 aberrations are more frequently observed in thick (15-μm) paraffin-embedded tissue sections by evaluating only complete nuclei. The use of routine sections (5-μm) for interphase cytogenetic analyses is compromised by a remarkable underestimation of the real chromosome copy numbers. Accepted: 7 November 1996