Decreased D2-40 immunoreactivity in stored paraffin sections and methods for preserving it

D2-40, a monoclonal antibody against podoplanin, is a selective marker of lymphatic endothelium and is widely used for research on and diagnosis of pathology of lymphatic vessels. We examined the relation between the duration of tissue section storage and changes in immunostaining by D2-40 antibody; we evaluated also the effects of preservation methods on changes in immunostaining during storage. Staining by D2-40 was attenuated by long-term preservation of scalp skin and lymph node sections at room temperature. The attenuation of D2-40 staining in stored sections was improved by preservation at low temperature, i.e., 4° or ? 30° C. We investigated also the immunostaining of preserved tissue sections using NZ-1 and Lyve-1, which are antibodies against lymphatic endothelium markers. Staining by NZ-1 or Lyve-1 antibody was detected clearly in sections that had been stored for 16 weeks. Our study suggests that either long-term storage of D2-40 immunostained tissue sections should be avoided or the section should be preserved at low temperature.     

Evaluation of hydrolytic enzyme activities from digestive fluids of Anthonomus grandis (Coleoptera: Curculionidae)

The cotton boll weevil, Anthonomus grandis, is a major pest of cotton crops in South America. In this work, partial biochemical characterizations of (hemi) cellulases and pectinases activities in the digestive system (head- and gut- extracts) of A. grandis were evaluated. Gut extract section from third instar larvae exhibited endoglucanase, xylanase, β-glucosidase, and pectinase activities. The endoglucanase and xylanase activities were localized in the foregut, whereas β-glucosidase activity was mainly detected in the hindgut. In addition, no difference in pectinase activity was observed across the gut sections. Thus, A. grandis digestive system is a potentially interesting reservoir for further lignocellulolytic enzymes research.     

Safranin and Anilin Blue with Delafield's Hematoxylin for Staining Cell Walls in Shoot Apexes

The following technic is suggested for staining cell walls in shoot apexes: After the usual preliminary steps through 50% ethyl alcohol, stain in 1 % safranin 0 for 24 hours. Rinse in tap water and place in 2% aqueous tannic acid for 2 minutes. After rinsing in tap water, stain for 2 minutes in 1 part Delafield's hematoxylin to 2 parts distilled water and rinse in tap water. Remove excess hematoxylin with acidified water (1 drop cone. HC1 in 200 ml. water), then place slides in 0.5% lithium carbonate for 5 minutes. Dehydrate through an ethyl alcohol series, then transfer from absolute alcohol to a saturated solution of anilin blue in “methyl cellosolve” for 5-10 minutes. Wash in absolute alcohol, rinse in a solution of 25% methyl salicylate, 33% xylene, 42% absolute ethyl alcohol and clear for 10 minutes in a solution of 2 parts methyl salicylate, 1 part xylene, 1 part absolute ethyl alcohol. Transfer through two changes of xylene and mount in “clarite” or suitable alternate. The resulting preparations will have clearly defined, dark-staining cell walls and will photograph well when “Super Panchro-Press, Type B” film (Eastman Kodak Co.) is used in conjunction with suitable Wratten filters.     

Amylase activity and growth in internodes of deepwater rice

Isoelectrofocusing, product analysis, thermal denaturation studies and affinity chromatography on cycloheptaamylose-Sephadex were used to identify the amylolytic enzymes in internodes of deepwater rice (Oryza sativa L.). Amylolytic activity in internodes of deepwater rice consists of -amylase (sometimes separated into two isoforms) and of -amylase. During submergence of whole plants, -amylase activity increases in young, growing internodes, but -amylase activity declines. Although non-growing, mature internodes contain higher levels of -amylase than do the elongating younger internodes, the effect of submergence on amylase activities in both tissues follows the same trend. Submergence, gibberellic acid (GA₃) and ethylene all promote -amylase activity in growing and non-growing internodes of excised deepwater-rice stem sections. Inhibitor studies showed that submergence and ethylene promote -amylase activity in the absence of endogenous gibberellin (GA), and GA₃ enhances -amylase activity when ethylene action is inhibited. Therefore, ethylene and GA appear to increase -amylase activity independently of each other. Enhanced -amylase activities are probably responsible for the mobilization of carbohydrates which are needed to support internode elongation during submergence of deepwater rice.Abbreviations CHA cycloheptaamylose - GA₃ gibberellic acid - NBD 2,5-norbornadiene - TCY tetcyclacis     

Fine structure of crystalline inclusions in B-cells of the islets of Langerhans in the alligator

Summary The ultrastructure of crystalline beta granules of the islets of Langerhans in the alligator has been investigated. From optical diffraction analysis and serial sectioning, the existence of four distinct types of crystalline inclusions was established in ultrathin sections. The first type is the most frequent and is interpreted as a rhombohedroni with a base, the ortho-hexagonal unit-cell edges being a=18.9 nm, c=23.0 nm. The second type of crystal (not observed in serial sections) is found compatible with a rhomb-dodecahedron which indexes on a cubic cell with a=9.6 nm. The third type of crystal was assigned to dipyramids. Dipyramids are extremely rare, and only two diffraction patterns were obtained; their crystal system could not be determined. Prisms, which are second in abundance, represent the fourth type of crystal. Spacings as well as the symmetry differ from those of the above three crystal types and indicate a tetragonal cell with a=4.2 nm, c=14.2 nm. The data for the prismatic crystals are strikingly similar to those of proinsulin and may represent the first case of agreement between crystals (i) formed in vitro and studied by X-ray diffraction and (ii) those investigated in situ by electron microscopy.     

A method for immunohistochemical detection of osteogenic markers in undecalcified bone sections

To evaluate the osteogenic potential of novel implant materials, it is important to examine their effect on osteoblastic differentiation. Characterizing the tissue response at the bone-biomaterial interface in vivo at a molecular level would contribute significantly to enhancing our understanding of tissue integration of endosseous implant materials. We describe here a new technique that overcomes difficulties commonly associated with performing immunohistochemistry on undecalcified sawed sections of bone. Sheep mandible specimens were fixed in an ethanol based fixative to maintain adequate antigenicity of the tissue. As a result, it was possible to omit antigen retrieval at high temperature for recovery of antigenicity, and detachment of sections from the slides was avoided. Following dehydration and infiltration, the specimens were embedded in a resin composed of polymethylmethacrylate and polybutylmethacrylate. Polymerization was achieved by adding benzoylperoxide and N,N-dimethyl-toluidine. This resin was selected because it maintained the antigenicity of the tissue, provided adequate properties for cutting 50 µm thick sections, and it facilitated deacrylizing the sawed sections. Acid-resistant acrylic slides were glued to the blocks using an epoxy resin based two-component adhesive to avoid detachment of the slides during the deacrylation procedure. Samples were stained for alkaline phosphatase, type I collagen, osteonectin, osteopontin, osteocalcin and bone sialoprotein. The EnVision + ™ dextran polymer conjugate two-step visualization system was applied for immunohistochemical detection of these bone matrix proteins. This procedure yielded positive staining for the osteogenic markers in cells and matrix components. The protocol described here facilitates the use of immunohistochemistry on resin embedded sawed sections of bone and provides a convenient and reliable method that can be used routinely for immunohistochemical analysis of hard tissue specimens containing implant materials.     

Maple phylogeny and biogeography inferred from phylogenomic data

Acer (the maple genus) is one of the diverse tree genera in the Northern Hemisphere with about 152 species, most of which are in eastern Asia. There are roughly a dozen species in Europe/western Asia and a dozen in North America. Several phylogenetic studies of Acer have been conducted since 1998, but none have provided a satisfactory resolution for basal relationships among sections of Acer. Here we report the first well‐resolved phylogeny of Acer based on DNA sequences of over 500 nuclear loci generated using the anchored hybrid enrichment method and explore the implications of the robust phylogeny for Acer systematics and biogeography. Our phylogenetic results support the most recent taxonomic treatment of Acer by de Jong with some modifications; section Pentaphylla may be expanded to include section Trifoliata, and A. yangbiense may be included in section Lithocarpa. Sections Spicata, Negundo, Arguta, and Palmata form a clade sister to the rest of the genus where sections Glabra and Parviflora comprise the first clade followed by section Macrantha, sections Ginnala, Lithocarpa, Indivisa, sections Platanoidea and Macrophylla, section Rubra, section Acer, and section Pentaphylla. Monotypic sections Glabra and Macrophylla in North America are sister to the Japanese section Parviflora and Eurasian section Platanoidea, respectively. Ancestral area inferences using statistical dispersal and vicariance analysis (S‐DIVA) and dispersal and extinction cladogenesis (DEC) methods suggest that Asia might be the most likely ancestral area of Acer as proposed by Wolfe and Tanai and molecular dating using Bayesian evolutionary analysis by sampling trees (BEAST) indicate that section diversifications of Acer might have completed largely in the late Eocene and the intercontinental disjunctions of Acer between eastern Asia and eastern North America formed mostly in the Miocene.     

Combined Lectin Binding and PAS/Alcian Blue Staining in Glycol Methacrylate Sections

Evaluation of cryofixation and paraffin and glycol methacrylate embedding showed that lectin binding was essentially independent of the embedding medium. Fluorescence intensity increased in the following order: glycol methacrylate, paraffin and cryostat sections, The optical resolution increased in the reverse order. Semi-thin glycol methacrylate sections provided satisfactory fluorescence intensities and the best resolution of all embedding techniques applied. Furthermore the lectin treated sections can be stained further using routine histological or specific histochemical methods. The potassium hy-droxide/alcian blue/periodic acid-phenylhydra-zine-Schiff method was used successfully to demonstrate sulfated and nonsulfated sialomucins. Lectins combined with mucin histochemistry allowed visualization of specific sugar residues in the same glycol methacrylate plastic section.     

Simplification of a light-based model for estimating final internode length in greenhouse cucumber canopies

Background and Aims

Light quantity and quality affect internode lengths in cucumber (Cucumis sativus), whereby leaf area and the optical properties of the leaves mainly control light quality within a cucumber plant community. This modelling study aimed at providing a simple, non-destructive method to predict final internode lengths (FILs) using light quantity and leaf area data.

Methods

Several simplifications of a light quantity and quality sensitive model for estimating FILs in cucumber have been tested. The direct simplifications substitute the term for the red : far-red (R : FR) ratios, by a term for (a) the leaf area index (LAI, m² m⁻²) or (b) partial LAI, the cumulative leaf area per m² ground, where leaf area per m² ground is accumulated from the top of each plant until a number, n, of leaves per plant is reached. The indirect simplifications estimate the input R : FR ratio based on partial leaf area and plant density.

Key Results

In all models, simulated FILs were in line with the measured FILs over various canopy architectures and light conditions, but the prediction quality varied. The indirect simplification based on leaf area of ten leaves revealed the best fit with measured data. Its prediction quality was even higher than of the original model.

Conclusions

This study showed that for vertically trained cucumber plants, leaf area data can substitute local light quality data for estimating FIL data. In unstressed canopies, leaf area over the upper ten ranks seems to represent the feedback of the growing architecture on internode elongation with respect to light quality. This highlights the role of this domain of leaves as the primary source for the specific R : FR signal controlling the final length of an internode and could therefore guide future research on up-scaling local processes to the crop level.