Histological investigation of organisms with hard skeletons: a case study of siliceous sponges.

Siliceous and calcareous sponges commonly are treated with acid to remove the spicules prior to embedding and cutting for histological investigations. Histology of spiculated sponge tissue represents a challenging problem in sponge histotechnology. Furthermore, fluorescence in situ hybridization (FISH), a key method for studying sponge-associated microbes, is not possible after acid treatment. For a broad range of siliceous sponge species, we developed and evaluated methods for embedding in paraffin, methylmethacrylate resins, LR White resin and cryomatrix. Different methods for cutting tissue blocks as well as mounting and staining sections also were tested. Our aim was to enable histological investigations and FISH without prior removal of the spicules. To obtain an overview of tissue and skeleton arrangement, we recommend embedding tissue blocks with LR White resin combined with en bloc staining techniques for large specimens with thick and numerous spicules, but paraffin embedding and subsequent staining for whole small specimens. For FISH on siliceous sponges, we recommend Histocryl embedding if the spicule content is high, but paraffin embedding if it is low. Classical histological techniques are used for detailed tissue examinations.     

Two Methods for Flat Embedding Sections in LR White Cut from Paraffin Blocks

Two simple methods are described for flat embedding of sections cut from paraffin blocks of brain tissue in the hydrophilic resin LR White. The ability of fresh resin to polymerize when added to already cured resin is exploited. In the first method, a tissue section with a drop of LR White is pressed to the bottom of a gelatin capsule using a blank block. In the second method, the section in a drop of resin is sandwiched between the sawed halves of a blank block. After curing, thin sections containing large areas of tissue can be collected and processed for immunocytochemistr y.     

Light inside sponges

Sponges are the most basal metazoan organisms. As sessile filter feeders in marine or freshwater habitats, they often live in close association with phototrophic microorganisms. Active photosynthesis by the associated microorganisms has been believed to be restricted to the outer tissue portion of the sponge hosts. However, phototrophic microorganisms have also been detected in deeper tissue regions. In many cases they are found around spicules, siliceous skelettal elements of demosponges and hexactinellids. The finding of phototrophic organisms seemingly assembled around spicules led to the hypothesis of a siliceous light transmission system in sponges. The principle ability to conduct light was already shown for sponge derived, explanted spicules. However it was not shown until now, that in deed sponges have a light transmission system, and can harbour photosynthetically active microorganisms in deeper tissue regions.Here we show for the first time, that, as hypothesized 13 year ago, sponge spicules in living specimens transmit light into deeper tissue regions. Our results demonstrate that in opposite to the actual opinion, photosynthetically active microorganisms can also live in deeper tissue regions, and not only directly beneath the surface, when a light transmission system (spicules) is present.Our results show the possibility of massive or globular sponges being supplied with photosynthetic products or pathways throughout their whole body, implying not only a more important role of these endobioses. Our findings also elucidate the in-situ function of a recently more and more interesting biomaterial, which is unique not only for its mechanical, electrical and optical properties. Biosilica is of special interest for the possibility to produce it enzymatically under environmental conditions.     

Evaluation of LR white resin for histology of the undecalcified rat tibia

Histology of plastic embedded undecalcified bone represents a challenging problem to the histotechnologist. We outline here an exploration of LR White resin as a suitable medium for histologic study of undecalcified rat tibia. A procedure was developed for light microscopy of rat tibia embedded in LR White and sectioned by sawing-grinding technics. The specimens were fixed in 10% neutral buffered formalin or alcohol-acetic acid-formol, dehydrated in ethanol, defatted in chloroform followed by resin infiltration and heat-curing of embedded blocks. The procedure of dehydration, defatting, infiltration, and polymerization can be completed within 10 days. Cold curing with accelerator provided by the manufacturer did not yield superior results compared to blocks cured with heat. Thick sections were obtained using a diamond wire saw, attached to plexiform slides, then ground and polished. Surface staining with Von Kossa silver reagent or toluidine blue revealed satisfactory morphological preservation of the mineralized bone sections. Artifacts like small bubbles appeared occasionally and could not be avoided despite prolonged infiltration or cold curing of blocks. Our method is relatively simple for base-line histologic study of rat tibia. The method offers advantages such as easy adaptability, reliable stainability, contrast, and resolution of bone architecture and marrow cells. Two other embedding media, Micro-Bed resin and Unicryl, were also tested, but produced inferior results.     

Evidence for spicule homology in calcareous and siliceous sponges: biminerallic spicules in Lenica sp. from the Early Cambrian of South China

Botting, J.P., Muir, L.A., Xiao, S., Li, X. & Lin, J.‐P. 2012: Evidence for spicule homology in calcareous and siliceous sponges: biminerallic spicules in Lenica sp. from the Early Cambrian of South China. Lethaia, Vol. 45, pp. 463–475. The relationships of the extant sponge classes, and the nature of the last common ancestor of all sponges, are currently unclear. Early sponges preserved in the fossil record differ greatly from extant taxa, and therefore information from the fossil record is critical for testing hypotheses of sponge phylogenetic relationships that are based on modern taxa. New specimens of the enigmatic sponge Lenica sp., from the Early Cambrian Hetang Biota of South China, exhibit an unusual spicule structure. Each spicule consists of a siliceous core with an axial canal, an organic outer layer and a middle layer interpreted to have been originally calcium carbonate. This finding confirms previous work suggesting the existence of biminerallic spicules in early sponges. Combined with data from other early sponges, the new findings imply that the two fundamental spicule structures of modern sponges were derived from a compound, biminerallic precursor. Spicules are therefore homologous structures in Calcarea and Silicea, and if sponges are paraphyletic with respect to Eumetazoa, then spicules may also have been a primitive feature of Metazoa. □Calcarea, Early Cambrian, Hetang Biota, phylogeny, Silicea, taphonomy.     

Use of LR white resin for post-embedding immunolabelling of brain tissue.

The object of this study was to investigate the applicability of the acrylic resin 'LR White' to immunolabelling of various antigenic determinants in aldehydefixed rat CNS tissue. Antibodies were used, which worked well in paraffin sections and therefore were suitable to detect antigens resistant to complete dehydration and heat. Different LR White embedding protocols were employed in order to select the preparation conditions that adequately preserved both the antigenicity and fine structure. Specimens were completely dehydrated with up to 100% ethanol, which was followed by various infiltration times with LR White monomer. Polymerization of the resin was induced by heat, a chemical catalytic procedure (accelerator), or ultraviolet (UV) light. Paraffin, as well as semithin and ultrathin LR White sections were incubated with antibodies reacting to antigens located on the cell surface (stage-specific embryonic antigen-1; SSEA-1), within the plasma membrane (myelin basic protein), in the cytosol (HNK-1, S100 protein), in the cytoskeleton (GFAP, vimentin, neurofilament protein, INT-FIL), and in the extracellular matrix (laminin). All of the examined antigens were immunocytochemically detectable in paraffin-embedded material, while the carbohydrate moieties, HNK-1 and SSEA-1, were not immunoreactive in LR White sections. However, in cryostat sections processed for pre-embedding immunoelectron microscopy, the HNK-1 epitope and SSEA-1 were immunolabelled. Polymerization carried out under UV light led to better structural preservation of brain tissue than resin cured with heat or catalyst. The length of prior infiltration with monomer apparently had no effect on tissue preservation. Consequently, UV light-induced polymerization of LR White gives acceptable morphology of brain tissue. However, the use of this acrylic resin is restricted to the detection of some CNS antigens only.     

Middle and Late Cambrian sponge spicules from Hunan, China

Abundant and well-preserved assemblages of disarticulated sponge spicules occur in Middle and Late Cambrian platform carbonates of western Hunan, China. Assemblages recovered from 11 stratigraphic horizons include calcisponges, demosponges, and hexactinellids. Hexactinellida, in particular, are both abundant and diverse in Upper Cambrian carbonates. Comparison with spicule assemblages from Australia indicates that many of these taxa have long stratigraphic ranges, limiting their use in correlation. The morphological diversity of these spicules exceeds that known for living siliceous sponges, supporting the observation that during the Cambrian radiation, sponges, like other metazoans, evolved a variety of architectural forms not observed in later periods. Like conodonts, individual sponges can produce more than one spicule form; thus, an "apparatus genus" concept based on multiple co-occurring elements may eventually prove useful in the biostratigraphic and paleobiological interpretation of disarticulated sponge spicules. Four distinctive forms are recognized as new taxa: Australispongia sinensis new genus and species, Flosculus gracilis new genus and species, Pinnatispongia bengtsoni new genus and species, and Nabaviella paibiensis new species.     

Calcification processes of siliceous sponges in Viséan Limestones (Counties Sligo and Leitrim,Northwestern Ireland)

Summary In the Lower Carboniferous limestones and shales of the Benbulben Range, Counties Sligo and Leitrim, northwestern Ireland, a suite of carbonate nodules, about 1 to 4 cm in diameter, has been sampled and investigated by thin sectioning. The nodules consist of micritic, peloidal and fenestral fabrics. Many of them contain relics of desma bearing demosponges and hexactinellid sponge skeletons. The nodules are interpreted as calcified siliceous sponges. Micrite and peloids have been formed via microbial activity during the decay of the soft sponge tissue. The actual processes are deduced from Recent examples investigated at Lizard Island, Autralia, byReitner (1993). The skeletal opal was dissolved very early. In places where the skeleton was already embedded in micrite the spicules are preserved as molds cemented by granular ferroan calcite. The nodules were extensively inhabited by agglutinating polychaetes and bored by sponges. Micrite clasts have been exported to the surrounding seafloor before the sponges were completely covered by sediments. Fenestral fabrics represent primary sponge cavities, that may be enlarged due to volume reduction of the soft tissue during calcification. Some originated from non-calcification of decaying tissue. The granular calcite cement, filling the fenestral fabrics, contains relics of spicules and faintly visible peloids floating unsupportedly in the cement. These peloids were probably produced in situ by calcification of organic mucilages that filled the cavities almost entirely. It is evident that most diagenetic processes occurring within the sponges happened on the seafloor, most likely within the still living individuals. Possibly the nodules represent a precursor stage of mud mound development.     

深海六放海绵大骨针的结构与特性

在海绵动物(多孔动物)中,六放海绵和寻常海绵为硅质骨骼.生活在深海(1 000 m)中的六放海绵是最古老的海绵动物,其中间单根海绵和春氏单根海绵有长达3 m的骨针,是地球上最长的生物硅结构.利用电子显微技术观测, 这些直径达8 mm的巨大根须骨针具有同心层状结构,其横截面显示明显的构造分界:中间为含有轴丝的轴管,外围是一50-150 μm厚的轴筒,最外面为区状区(300-500层,每层厚度3-5 μm).生物化学研究显示其主要的蛋白质为35 kD大分子,另外,还检测到23-24 kD 多肽,可能是硅蛋白相关蛋白.依据现有的红血球凝聚活性,从骨针提取物中也检测到了凝集素.由电子探针获得其化学成分主要为Si,K和Na.此外,骨针的光传输实验表明,该巨大根须骨针用作光纤可传输600 nm至1 400 nm范围的光,而滤掉小于600 nm的光(类似高通滤波器)和大于1 400 nm 的红外光(类似低通滤波器).另外,从六放海绵的空囊泡沫海绵中分离出一个基因并确证了其推导的编码蛋白序列,该蛋白编码一个光裂合酶相关蛋白,蛋白相似性比较结果显示属于光裂合酶相关蛋白中多细胞动物隐色素一类.基于以上数据给出了六放海绵硅质骨针形成的示意图.另外,由单根海绵骨针可作为波导传输光/电和/或化学信号,推断在海绵动物中有类似神经系统的网络系统[动物学报 53(3):557-569,2007].     

Effects of long-term fixation on histological quality of undecalcified murine bones embedded in methylmethacrylate.

While long-term fixation and storage of specimens is common and useful for many research projects, it is particularly important for space flight investigations where samples may not be returned to Earth for several months (International Space Station) or years (manned mission to Mars). We examined two critical challenges of space flight experimentation: the effect of long-term fixation on the quality of mouse bone preservation and the preservation of antigens and enzymes for both histochemical and immunohistochemical analyses, and how the animal/sample processing affects the preservation. We show that long-term fixation minimally affects standard histological staining, but that enzyme histochemistry and immunolabeling are greatly compromised. Further, we demonstrate that whole animal preservation is not as suitable as whole leg or stripped leg preservation for long-term fixation and all histological analyses. Overall, we recommend whole leg processing for long-term storage of bone specimens in fixative prior to embedding in plastic for histological examination.     

Effects of long-term fixation on histological quality of undecalcified murine bones embedded in methylmethacrylate

While long-term fixation and storage of specimens is common and useful for many research projects, it is particularly important for space flight investigations where samples may not be returned to Earth for several months (International Space Station) or years (manned mission to Mars). We examined two critical challenges of space flight experimentation: the effect of long-term fixation on the quality of mouse bone preservation and the preservation of antigens and enzymes for both histochemical and immunohistochemical analyses, and how the animal/sample processing affects the preservation. We show that long-term fixation minimally affects standard histological staining, but that enzyme histochemistry and immunolabeling are greatly compromised. Further, we demonstrate that whole animal preservation is not as suitable as whole leg or stripped leg preservation for long-term fixation and all histological analyses. Overall, we recommend whole leg processing for long-term storage of bone specimens in fixative prior to embedding in plastic for histological examination.     

Block Staining of Mammalian Tissues with Hematoxylin and Eosin

Various mammalian tissues were stained en bloc with hematoxylin and eosin after fixation and prior to embedding in paraffin wax and sectioning. The choice of fixative is important and best results are obtained using Worcester's Fluid, a combination of saturated aqueous mercuric chloride, formaldehyde, and glacial acetic acid. After fixation, blocks of tissue up to 1.5 cm thick are stained for seven days in hematoxylin. Excess stain is removed by washing tissues in running water overnight. Tissue blocks then are dehydrated with graded concentrations of ethyl alcohols to 80% and counterstained, with further dehydration, in 0.5% spirit soluble eosin in 90% ethyl alcohol for five days. The tissue is subsequently transferred to 90% ethyl alcohol overnight to differentiate eosin staining; dehydration is completed in absolute ethyl alcohol. The blocks are cleared in cedarwood oil and briefly in xylene prior to embedding, sectioning, and mounting. Following removal of wax by xylene, coverslips are applied.

General morphological and histological features were particularly well differentiated and very selectively and reliably stained by this method.     

Block staining of mammalian tissues with hematoxylin and eosin

Various mammalian tissues were stained en bloc with hematoxylin and eosin after fixation and prior to embedding in paraffin wax and sectioning. The choice of fixative is important and best results are obtained using Worcester's Fluid, a combination of saturated aqueous mercuric chloride, formaldehyde, and glacial acetic acid. After fixation, blocks of tissue up to 1.5 cm thick are stained for seven days in hematoxylin. Excess stain is removed by washing tissues in running water overnight. Tissue blocks then are dehydrated with graded concentrations of ethyl alcohols to 80% and counterstained, with further dehydration, in 0.5% spirit soluble eosin in 90% ethyl alcohol for five days. The tissue is subsequently transferred to 90% ethyl alcohol overnight to differentiate eosin staining; dehydration is completed in absolute ethyl alcohol. The blocks are cleared in in cedarwood oil and briefly in xylene prior to embedding, sectioning, and mounting. Following removal of wax by xylene, coverslips are applied. General morphological and histological features were particularly well differentiated and very selectively and reliably stained by this method.     

Does the skeleton of a sponge provide a defense against predatory reef fish?

Sponge tissue often contains two structural components in high concentrations: spicules of silica, and refractory fibers of protein (spongin). Some terrestrial plants contain analogous structures, siliceous inclusions and refractory lignins, that have been demonstrated to deter herbivory. We performed feeding experiments with predatory reef fish to assess the deterrent properties of the structural components of three common Caribbean demosponges, Agelas clathrodes, Ectyoplasia ferox, and Xestospongia muta. The concentrations of spicules and spongin in the tissues varied widely between the three species, but when assayed at their natural volumetric concentrations, neither spicules (all three species assayed) nor the intact spiculated spongin skeleton (A. clathrodes and X. muta assayed) deterred feeding by reef fish in aquarium or field assays using prepared foods of a nutritional quality similar to, or higher than, that of sponge tissue. Spicules deterred feeding in aquarium assays when incorporated into prepared foods of a nutritional quality lower than that of sponge tissue (15–19 times less protein), but spiculated spongin skeleton was still palatable, even in prepared foods devoid of measurable protein, and even though spicules embedded in spongin were oriented in their natural conformation. Based on comparisons of the nutritional qualities of the tissues of the three sponge species and of the prepared foods, sponge tissue would have to be much lower in food value (5 times less protein or lower) for spicules to provide an effective defense, and spicules in combination with the spongin skeleton would be unlikely to provide an effective defense regardless of the nutritional quality of the tissue. Unlike terrestrial plants, marine sponges may use silica and refractory fibers solely for structural purposes.     

Spicules of hexactinellid sponges (Hexactinellida: Porifera) as natural composite materials

This paper reviews studies on the hexactinellid glass sponges (Hexactinellida: Porifera) that have organic silica spicules. According to its physical properties (microdensity, Young’s modulus, and light transmission), the material of the spicules is similar to amorphous silica; however, sponge spicules are birefringent, which suggests that they have a highly ordered crystal-like nature. Mineralized remnants of siliceous spicules composed of chemically inert materials are preserved in sedimentary rocks and provide evidence of the ecological state of the ancient biosphere. Sponges occur in waters with low temperatures; therefore, they grow very slowly and live for hundreds of years. The organic silica spicules exhibit the capacity for triboluminescence. The generated light emission may be used by symbiotic bacteria on the spicule surface.     

Processing of immunoisolated pancreatic islets: implications for histological analyses of hydrated tissue

Routine tissue processing is usually associated with histological artifacts as a consequence of shrinkage and distortion during dehydration required for embedding. With hydrated specimens such as lung, embryonic, and tissues in hydrophilic membranes, tissue processing can induce severe artifacts that interfere with adequate microscopic evaluation. Here we present a method for embedding hydrophilic alginate-polylysine microencapsulated pancreatic tissue that combines the absence of histological artifacts with a practical tissue processing method. We found that the glycol-methacrylate (GMA)-embedding method preserved the integrity of the encapsulated tissue better than snap-freezing or paraffin embedding, but the overall quality of the hydrophilic capsules remained poor Next, we modified the GMA method by introducing gradual dehydration to investigate whether the integrity of the sectioned capsules was better maintained by a more gradual pattern of water extraction. This modification resulted in well-preserved morphological details of the hydrophilic membranes, hydrogel-cell interface, and encapsulated pancreatic tissue. Subsequent routine staining gave excellent contrast between the islet tissue and hydrophilic components, which allowed adequate quantitative histological and pathological comparisons.     

The caddisfly Ceraclea fulva and the freshwater sponge Ephydatia fluviatilis: a successful relationship

The association between the aquatic phases of the caddisfly Ceraclea fulva (Trichoptera, Leptoceridae) and the freshwater sponge Ephydatia fluviatilis (Porifera, Spongillidae) has been investigated by means of scanning and transmission electron microscopy (SEM, TEM). Ceraclea fulva habitually feeds on sponges and builds its case by using the siliceous spicules of the sponge, which are arranged side by side, inter-crossed, cemented with silk, and organised in layers. In the newly hatched larva, the case is strengthened exclusively by cemented siliceous spicules, while during growth, the insect adds sponge fragments to it. The fine organisation of the sponge tissues growing on the case proves that the sponge is functional. Inter-spaced, small protrusions, derived from the outermost compact silk layer, form a series of "bridges" enhancing case/sponge adhesion. Tube-case shape varies according to the aquatic developmental phase of the insect: in the mature larva and pupa, this shelter carries larger sponge fragments dorsally. The caddisfly acts as carrier of the sponge, thus facilitating its dispersal and the colonisation of new habitats. This justifies regarding this association as a successful mutualistic relationship, and not as a unilateral parasitic behaviour on the part of the insect.     

Histological Techniques to Improve Testing of Pulpal Response of Teeth to Filling Material

Two fixation fluids, two fixation techniques and two embedding methods were investigated for their effects on the quality of sections of teeth for pulpal response to filling materials to improve evaluation of pulpal responses. Sections from 32 baboon teeth were prepared, half with experimental cavities and half without, using either 10% formaldehyde or 4% glutaraldehyde, longitudinal tooth splitting or removal of the tooth apex, and paraffin or K plast resin embedding; decalcification in a formic acid mixture was a constant throughout. Histometric analysis showed that paraffin embedding produced less shrinkage than the K Plast resin embedding although the difference was not statistically significant. Six parameters of separation at the pu1p:dentine interface were studied: embedding, fixative, presence or absence of a cavity, cutting technique and individual animal tooth type. Statistical investigation revealed that fixative, cutting technique, and fixative and cutting technique combined had significant influences on the separation artifact. Of the combinations tested the choice of embedding method depends on which of the two artifacts, shrinkage or separation, is more adverse in the opinion of the investigator. Four percent glutaraldehyde together with the longitudinal split technique of fixation. processed by either K Plast resin embedding or paraffin embedding produced satisfactory pulpal sections.     

Applicability of using acrylic resins in post-embedding ultrastructural immunolabelling of human neutrophil granule proteins

Summary In this study, quantitative assessments were carried out, (1) by light microscopy during tissue preparation for electron microscopy and (2) by electron microscopy after on-grid immunogold staining, to determine the suitability of using LR White and Lowicryl K4M thin sections to identify lactoferrin and elastase in the granules of human neutrophil leucocytes. Quantitative assessment of the effect of fixation, dehydration and embedding on the preservation of antigenicity during tissue preparation for electron microscopy, using light microscopic peroxidase anti-peroxidase immunocytochemistry, enabled the selection of preparation conditions that adequately preserved both antigenicity and ultrastructure. OsO₄ post-fixation, following primary aldehyde fixation, improved the retention of antigenicity during dehydration and embedding and the preservation of fine structure. Partial rather than complete dehydration retained more of the antigenicity. The efficiency, sensitivity and resolution of immunolabelling and the ultrastructure and quality of sections achieved after embedding in LR White were superior to those obtained after embedding in Lowicryl K4M. Consequently room temperature embedding in LR White following double fixation and partial dehydration is a better and more reliable preparation technique than low-temperature embedding in Lowicryl K4M following single fixation and partial dehydration for localizing lactoferrin and elastase to the specific and primary granules respectively in human neutrophilic granulocytes by the on-grid immunogold staining method.     

The morphological and immunohistochemical analysis of renal biopsies by light and electron microscopy using a single processing method

A methodology is described in which a number of well-established research techniques are brought together to enable the complete diagnostic analysis of a renal biopsy on a single piece of tissue. By embedding the biopsy in the acrylic resin LR White, unsupported sections of which are stable in the electron beam, light and electron microscopy and immunocytochemistry become feasible on sections from the same block. The biopsy is glutaraldehyde fixed but post-fixation in osmium tetroxide, which is often deleterious to antigen preservation, is omitted. Extraction in organic solvents and resin monomer is minimized by rapidly infiltrating the tissue from 70% ethanol and polymerizing the resin catalytically at 0 degrees C. Semithin sections can be stained with haematoxylin and eosin, Toluidine Blue or methenamine silver, giving results similar or superior to those obtained from paraffin sections. Thin sections show that the standard of morphological preservation is similar to that seen using epoxide sections even though the kidney is unosmicated. The tissue retains a high level of antigen reactivity, which, in the limited number of cases so far examined, has paralleled or exceeded that demonstrated by conventional immunofluorescence on frozen sections.