A Method for Histological Examination of Undecalcified Teeth

A method is presented for histological examination of undecalcified ground sections of tooth roots affected with periodontal disease. The roots were placed in Karnovsky's fixative overnight, postfixed in 2% buffered osmic acid, and dehydrated in an ascending series of ethanol. The specimens were then infiltrated with propylene-oxide and Epon-Araldite resin, embedded in Epon-Araldite, and sections were prepared using a cutting and grinding system. The resulting ground sections were 8-12 μm thick. The sections were allowed to air dry at room temperature. When thoroughly dried, a coverglass was applied using resinous mounting medium DPX. The specimens were examined by phase-contrast microscopy. The method is useful for simultaneous examination of mineralized dental tissue and bacterial morphotypes covering the root surface of teeth involved with periodontal disease.     

Removal of histological sections from glass for electron microscopy: use of Quetol 651 resin and heat

A method is described for using the epoxy resin Quetol 651 and heat for convenient and rapid separation of conventional histological sections from glass slides for subsequent ultrathin sectioning for retrospective electron microscopy. The same method is useful when Epon-Araldite is substituted for the Quetol 651 resin.     

Removal of Histological Sections from Glass for Electron Microscopy: Use of Quetol 651 Resin and Heat

A method is described for using the epoxy resin Quetol 651 and heat for convenient and rapid separation of conventional histological sections from glass slides for subsequent ultrathin sectioning for retrospective electron microscopy. The same method is useful when Epon-Araldite is substituted for the Quetol 651 resin.     

Bromodeoxyuridine and lododeoxyuridine Double Immunostaining for Epoxy Resin Sections

To establish bromodeoxyuridine (BrdUrd)/iododeoxyuridine (IdUrd) double immunostaining for thick sections of epoxy resin-embedded tissues, young hamsters received intra-peritoneal injections of IdUrd and BrdUrd 3 hr and 1 hr before sacrifice, respectively. The intestines were fixed with phosphate-buffered 4% paraformaldehyde and embedded in an Epon-Araldite mixture. The epoxy resin was removed completely by a sodium methoxide/benzene/methanol solution. This epoxy resin removal method was effective for BrdUrd/IdUrd immunostaining using a mono-specific antibody for BrdUrd (Br-3) and a bi-specific antibody for BrdUrd and IdUrd (IU-4), followed by the ABC complex method. Epoxy sections stained with these antibodies showed clear localization of nuclei incorporating the two thymidine analogues with precise morphology of labeled cells. Furthermore, ultrastructural observation of thin sections adjacent to thick sections immunostained for BrdUrd/IdUrd confirmed the cell type and ultrastructural features of cells labeled with these thymidine analogues.     

Evaluation of Preparation, Staining and Microscopic Techniques for Counting Incremental Lines in Cementum of Human Teeth

Apposition of cementum occurs in phases resulting in two types of layers with different optical and staining properties that can be observed by light microscopy. Narrow, dark staining incremental lines are separated by wider bands of pale staining cementum. The distance from one line to the next represents a yearly increment deposit of cementum in many mammals, and counting these lines has been used routinely to estimate the age of the animals. Incremental lines in cementum have also been observed in sections of human teeth, and the object of the present investigation was to examine a number of methods for preparing and staining them for counting. Longitudinal and transverse sections, either ground or decalcified, were cut from formalin fixed human dental roots, paraffin embedded or frozen, and stained using several techniques. The cementum was investigated using conventional light, fluorescence, polarized light, confocal laser scanning, interference contrast, phase contrast, and scanning electron microscopy. Incremental lines in the cementum could be observed in ground sections and, following decalcification, in both frozen and paraffin embedded sections. Toluidine blue, cresyl violet, hematoxylin, or periodic acid Schiff (PAS) stained incremental lines allowing differentiation by conventional light microscopy. Contrast was best using fluorescence microscopy and excitation by green light since the stained cemental bands, but not the incremental lines, fluoresced after staining with cresyl violet, PAS or hematoxylin and eosin. The results with other microscopic techniques were unsatisfactory. Since incremental lines are not destroyed by acids and stain differently than the remaining cementum, it is likely that they possess an organic structure which differs from the cementum. Incremental lines in human dental cementum could be observed best using decalcified sections stained with cresyl violet excited by green light.     

植入了骨修复材料的小型猪腰椎椎体不脱钙病理组织切片制备方法

摘要 目的：建立植入了骨修复材料小型猪腰椎椎体骨组织标本的不脱钙病理组织切片制备方法。方法：将含骨修复材料的腰椎椎体骨组织标本进行分割暴露组织切面,梯度浓度乙醇脱水后经Technovit 7200 VLC光聚树脂浸润,经黄蓝光共同辐照进行光聚合包埋,借助硬组织病理切磨系统制备含骨修复材料不脱钙病理组织切片。结果：结果显示通过上述方法制备的病理组织切片,经苏木精-伊红（HE）染色及甲苯胺蓝染色后光学显微镜下观察能较好地显示骨的各种组织细胞结构,可清晰的观察到骨小梁的走向及连接情况。结论：研究建立了含骨修复材料骨组织标本病理组织切片制备方法,实现了含骨修复材料不脱钙骨组织病理切片的制备,经病理染色后实现了带植入物的组织学观察,为生物材料及医疗器械动物试验研究提供了新的病理检测手段及组织学评价途径。     

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.     

Embedding Thin Plant Specimens for Oriented Sectioning

Small plant structures such as small primary roots, filamentous mosses and algae are difficult to orient for sectioning since they become wavy and curl during embedding. A method is described for embedding and orienting tiny plant specimens in a glycol methacrylate resin using self-constructed flat molds. Prior to sectioning, small samples can be oriented in both the longitudinal and the transverse plane. As several samples can be sectioned simultaneously, time-consuming trimming of the blocks is reduced substantially. The efficiency of this technique has been demonstrated using the tiny roots of the model plant Arabidopsis thaliana (L.) Heynh.     

Applications of a novel biodetection system to saliva using protein fingerprints with data processing

A fundamental method has been developed focusing on a facile and rapid examination of periodontal disease. Periodontal disease is an oral disease thought to affect 80% of adults, and early detection with treatment is desirable for the improvement of the quality of life. Unfortunately conventional methods are not consistent as the disease is caused by a number of undefined bacteria and detection relies on the skills of the dentist. Thus an objective detection system is required. We have performed an experiment on saliva using a novel biodetection system, designated PepTenChip®. A disease model for saliva was prepared using a specimen from a healthy subject and a mixture of hemoglobin (f-Hb) and lactate dehydrogenase (LDH), which is used as a periodontal disease marker protein with healthy saliva. PepTenChip® is a peptide microarray in which fluorescent labelled structured peptides are immobilized on a novel amorphous carbon substrate. Since the peptides used as capture molecules are fluorescently labelled, labeling of analytes is not necessary. The fluorescence intensity change before and after application of analytes are detected rather than the ON/OFF detection common to conventional microarrays using a set of antigen–antibody. The fluorescence intensity value changes according to the concentration of captured protein allowing the generation of protein fingerprint (PFP) and dendrograms. The present method does not rely on a “one to one” interaction, unlike conventional biodetection, and advantages can be envisaged in the case of an undefined or unknown cause of disease. The statistical analyses, such as multivariate analyses, allow classification of the type of proteins added in saliva as mimetics of disease. PepTenChip® system is useful and convenient for examination of periodontal disease in health care.     

Preparation of whole rabbit knee joints for microscopic examination

A paraffin embedding method to prepare whole rabbit knee joints for histological examination is described. This method provides good quality microscopic sections thin enough for the study of cellular detail and does not require prolonged processing. When examining pathologic changes in experimental arthritis, it is advantageous to be able to examine the intact joint with the structural relations of the joint components preserved. Sections of the whole joint provide numerous areas where bone, cartilage and synovium are contiguous for examination. Having obtained poor results using methods recommended for small bony specimens, we modified several existing procedures to obtain a reliable method for preparing excellent microscopic sections of the whole rabbit knee joint.     

Preparation of Whole Rabbit Knee Joints for Microscopic Examination

A paraffin embedding method to prepare whole rabbit knee joints for histological examination is described. This method provides good quality microscopic sections thin enough for the study of cellular detail and does not require prolonged processing. When examining pathologic changes in experimental arthritis, it is advantageous to be able to examine the intact joint with the structural relations of the joint components preserved. Sections of the whole joint provide numerous areas where bone, cartilage and synovium are contiguous for examination. Having obtained poor results using methods recommended for small bony specimens, we modified several existing procedures to obtain a reliable method for preparing excellent microscopic sections of the whole rabbit knee joint.     

Macropore sheath: quantification of plant root and soil macropore association

Plants require roots to supply water, nutrients and oxygen for growth. The spatial distribution of roots in relation to the macropore structure of the soil in which they are growing influences how effective they are at accessing these resources. A method for quantifying root-macropore associations from horizontal soil sections is illustrated using two black vertisols from the Darling Downs, Queensland, Australia. Two-dimensional digital images were obtained of the macropore structure and root distribution for an area 55 × 55 mm at a resolution of 64 μm. The spatial distribution of roots was quantified over a range of distances using the K-function. In all specimens, roots were shown to be clustered at short distances (1–10 mm) becoming more random at longer distances. Root location in relation to macropores was estimated using the function describing the distance of each root to the nearest macropore. From this function, a summary variable, termed the macropore sheath, was defined. The macropore sheath is the distance from macropores within which 80% of roots are located. Measured root locations were compared to random simulations of root distribution to establish if there was a preferential association between roots and macropores. More roots were found in and around macropores than expected at random. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ultrastructural immunogold labelling of vimentin filaments on postembedding ultrathin sections of arachnoid villi and meningiomas

An immunoelectron microscopic technique for the labelling of vimentin intermediate filaments on postembedding ultrathin sections is reported. Arachnoid villi obtained at autopsy and meningiomas at surgery were fixed in 1% paraformaldehyde for 30 minutes, embedded without postfixation in Epon-Araldite mixture and polymerized at 37 degrees C for 3 weeks. Ultrathin sections were etched in 2% KOH for 3 minutes and incubated with anti-vimentin monoclonal antibodies which were subsequently labelled with goat anti-mouse IgG coupled to colloidal golds. All of these labelling procedures were consistently performed within 4 hours. In both arachnoidal and meningioma cells, immunogolds preferentially decorated the intermediate filaments in proportion to the concentration. Very few gold particles were seen over the nucleus, Golgi zone, mitochondria and the extracellular connective tissue fibres. The present technique may be applied to the immunogold labelling of intermediate filaments on postembedding ultrathin sections.     

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.     

Optimization of a histopathological biomarker for sphingomyelin accumulation in acid sphingomyelinase deficiency

Niemann-Pick disease (types A and B), or acid sphingomyelinase deficiency, is an inherited deficiency of acid sphingomyelinase, resulting in intralysosomal accumulation of sphingomyelin in cells throughout the body, particularly within those of the reticuloendothelial system. These cellular changes result in hepatosplenomegaly and pulmonary infiltrates in humans. A knockout mouse model mimics many elements of human ASMD and is useful for studying disease histopathology. However, traditional formalin-fixation and paraffin embedding of ASMD tissues dissolves sphingomyelin, resulting in tissues with a foamy cell appearance, making quantitative analysis of the substrate difficult. To optimize substrate fixation and staining, a modified osmium tetroxide and potassium dichromate postfixation method was developed to preserve sphingomyelin in epon-araldite embedded tissue and pulmonary cytology specimens. After processing, semi-thin sections were incubated with tannic acid solution followed by staining with toluidine blue/borax. This modified method provides excellent preservation and staining contrast of sphingomyelin with other cell structures. The resulting high-resolution light microscopy sections permit digital quantification of sphingomyelin in light microscopic fields. A lysenin affinity stain for sphingomyelin was also developed for use on these semi-thin epon sections. Finally, ultrathin serial sections can be cut from these same tissue blocks and stained for ultrastructural examination by electron microscopy.     

Rapid Three-Dimensional Reconstruction at the Light Microscopic Level and a Technique for Re-Embedding the Same Semithin Sections for Electron Microscopic Examination

Rapid three-dimensional reconstruction of serial sections at the light microscopic and ultrastructural levels was accomplished using a two-step technique. Fixed specimens were embedded in Epon and 1 μm sections were cut and placed on glass slides. One of every four sections was drawn onto transparency film for rapid three-dimensional reconstruction. The semi-thin sections were re-embedded in Epon and sectioned at 90 nm for examination in the electron microscopy.     

A method for obtaining histological sections from tissue previously studied by scanning electron microscopy

An accelerated method of paraffin embedding of tissue specimens previously examined with scanning electron microscopy is proposed aimed to obtain sections for routine histological examination. The tissue is passed through acetone, absolute alcohol, alcoholic-oil celloidin solution, chloroform to be eventually mounted into paraffin. The method allows obtaining good quality sections within 24 hours.     

A staining method using acridine orange and auramine O for fungi and mycobacteria in bovine tissue.

A rapid staining method using auramine O and acridine orange (AOAO) is described for staining mycobacteria and fungi in paraffin sections of bovine tissues. One hundred seventy-seven tissue sections from specimens divided into two general groups on the basis of previous histopathology results were examined with a fluorscent microscope. Group I, a total of 77 sections, were from 47 mycobacterial and 30 fungal granulomas. Mycobacteria were found by the AOAO procedure in 44 of the 47 tissues previously diagnosed as positive for mycobacteria. All 30 fungal granulomas previously diagnosed using convential fungal stains were positive with the AOAO procedure. Group II consisted of sections prepared from 100 granulomas in which typical mycobacterial lesions were observed by histopathologic examination but in which no mycobacteria had been detected. Using the AOAO procedure, two of these 100 granulomas were found to contain mycobacteria and two were found to contain bacterial colonies. In the remaining 96 no etiologic agent could be demonstrated.     

A mass collection of Triatoma ryckmani (Hemiptera: Reduviidae) from Stenocereus Eichlamii (Cactaceae) in the semiarid region of Guatemala

A population of 216 specimens of Stenocereus eichlamii (Cactaceae, Subfamily Cereoideae) was surveyed for Triatoma ryckmani (vector of Chagas disease) in a one hectare plot of semiarid habitat in Guatemala. Out of 44 plants that had dead and dry sections, 24 plants had a total of 103 specimens of T. ryckmani. In comparison with other areas of Guatemala, T ryckmani is well established in the semiarid ecosystem (Infestation index 54.5, density = 2.3 and crowding index 4.3). The insects were mainly found (52.4%) in the dead portions of S. eichlamii 2.0 to 3.2 m above the ground (X2 = 26.0, P < 0.00001), followed by dry cactus sections between 3.2 and 5.0 m (35.9%). They were less frequent 0.2 to 2.0 m above the ground. A considerable proportion (75.7%) had no aparent blood in their digestive systems. To determine the presence of flagellates, 43 of the bugs were dissected, but none were found. This is the first report on T. ryckmani population dynamics in this habitat.