bluuebunny/crossref_metadata_2025_split huggingface dataset:
How the fixation‐embedding protocol affects the specificity and efficiency of immunocytochemical stains for gonadotropin subunits
Authors: Childs Gwen V. , Unabia Geda , Tibolt Robert 
Published: February 2005

AbstractThis report describes a study designed to test factors that may affect the efficiency and specificity of stains for gonadotropins. These include chemical or freeze‐fixation and dehydration, heat polymerization of the plastic embedding compound, dehydration in organic solvents, and etching. Specifically, postembedding stains for LH or FSH subunits were applied to 1‐μm sections of (1) Araldite‐embedded pituitaries that were either chemically fixed and dehydrated or freeze‐fixed and freeze‐dried; (2) Aldehyde‐fixed pituitaries that were dehydrated in water‐soluble glycol methacrylate (GMA) and embedded in GMA at 4°C; and (3) p‐formaldehyde‐fixed pituitaries that were embedded in paraffin. A fourth group of pituitaries was dispersed and grown in monolayers for 1–3 days. These were stained following glutaraldehyde fixation. The optimal dilution of the primary antisera varied with the protocol; however, the percentage of cells staining for beta subunits did not change. In contrast, postembedding stains showed that alpha subunit reactivity is masked or destroyed in pituitaries that are fixed in glutaraldehyde and embedded in Araldite. Alpha chain reactivity was detected (in 14% of cells) either after freeze‐fixation and freeze‐drying followed by Araldite embedding, or after 4% paraformaldehyde fixation and GMA embedding (in 17% of cells). Staining in paraffin‐embedded pituitaries was seen in only 10% of the cells. Preembedding stains for alpha chains were strikingly sensitive, however, and immunoreactivity was seen in 18–26% of the population of monolayer cells. Thus, whereas the percentages of cells staining for beta subunits do not change following the use of most of the fixation and embedding protocols, alpha chain reactivity is destroyed by all but the mildest. These findings show that one can control or improve the specificity of the stains for LH and FSH by the fixation‐embedding protocol.
Observations on the solitary cilium of rabbit oviductal epithelium: Its motility and ultrastructure
Authors: Odor D. Louise , Blandau Richard J. 
Published: February 2005

AbstractSolitary cilia have been observed on rabbit oviductal epithelial cells. In tissue cultures of fimbrial epithelium of 3‐ and 4‐day‐old animals observed by phase microscopy, most of these single cilia exhibited a vortical or funnel‐type movement while others had the usual to‐and‐fro motility. Primary cilia are usually considered immotile. Transmission electron microscopy of specifically identified single cilia revealed differences between the ciliary shafts and basal bodies of the single cilia as compared to those of mature oviductal ciliated cells. The basal body of the solitary cilium often had at least two triangular, striated, basal foot processes, lacked electron‐dense satellite material around its basal end, and occasionally had striated rootlets. In contrast, the cilia of mature ciliated cells had only one basal foot, exhibited much electron‐dense satellite material, and lacked rootlets. Cross sections of the single cilia showed patterns of microtubules different from the usual 9 + 2 axonemal complexes of normal cilia and included 9 + 0, 10 + 2 singlets, 7 + 2 doublets, and 8 + 1 doublet and 2 singlets; one did have the usual 9 + 2 arrangement. We postulate that the presence of more than one basal foot process may be responsible for the vortical motility observed. The primary cilia are shorter than normal cilia; the longest one measured was 1.86 μm in length, 0.28 μm in width at its base, and 0.14 μm at its tip. Based on the lightmicroscopic, scanning‐electron‐microscopic and transmission‐electron‐microscopic observations, such solitary cilia were observed more frequently in the oviductal tissues of the 3‐ to 4‐day postnatal rabbits grown in tissue culture and in ovariectomized and ovariectomized/progesterone‐treated adult animals than in estrous, ovulatory, or ovariectomized/estradiol‐treated rabbits.
Immunocytochemistry: Its evolution and criteria for its application in the study of epon‐embedded cells and tissue
Authors: Gosselin Edmund J. , Cate Charles C. , Pettengill Olive S. , Sorenson George D. 
Published: February 2005

AbstractThe word immunocytochemistry is currently used to describe a number of methods that can be employed to localize antigens within cells by means of antigen‐specific antibodies. In this article we will review a number of these methods, including immunofluorescence, immunoperoxidase, avidinbiotin, and colloidal‐gold techniques. The advantages and disadvantages of the various methods are discussed, special attention being focused upon immunocytochemical staining of plastic‐embedded tissue. Studies on the light microscope level show that embedding tissue in plastic prior to immunoperoxidase staining not only improves visualization of antigen‐specific staining but also provides an accurate and efficient means of prescreening tissue for antigen prior to immunocytochemical staining on the electron microscope level. Varying section thickness between 1 and 3 μm does not significantly influence staining, whereas the fixative used to preserve the tissue under study does. On the electron microscope level, the colloidal gold technique appears superior to immunoperoxidase staining. It is both esthetically more pleasing and highly sensitive. Of five different colloidal gold methods tested, the most sensitive is the two‐step technique that employs an antigen‐specific primary antibody followed by a gold‐labeled secondary antibody. Throughout this article, special emphasis is placed on the use of proper controls, both on the light and electron microscope levels. Where possible, such controls should include (1) substitution of specific antiserum with normal serum; (2) the use of antigen‐adsorbed antiserum; (3) the use of antisera with specificities for antigens not present in the tissue being studied; (4) the use of tissue previously shown to be stainable for the antigen; and (5) if cultured cells are being studied, the use of a number of cell types that do not contain the antigen.
Use of pre‐embedding ultrastructural immunocytochemistry in the localization of a secretory product and membrane proteins in cultured prolactin cells
Authors: Tougard Claude , Picart Renée 
Published: February 2005

AbstractA pre‐embedding immunoperoxidase procedure performed directly on cultured cells in situ was used to localize several intracellular antigens at the electron‐microscope level. With this procedure, we compared the effect of various fixatives, with or without saponin permeabilization, on the immunoreactivity of a secretory product (prolactin) and membrane proteins in cultured prolactin cells. Prolactin was detected within all compartments of its intracellular secretory pathway. Membrane antigens of the rough endoplasmic reticulum, the Golgi apparatus, and lysosomes were localized in distinct intracellular compartments. These immunocytochemical results are discussed in relationship to others in the literature that describe the localization of similar types of antigens. The technique, here described, which preserves ultrastructural detail and antigenicity, should be applicable for the localization of other intracellular antigens in cultured cells.
Combined light and electron microscope immunocytochemical localization of scattered peptidergic neurons in the central nervous system
Authors: Anthony Edythe L. P. , King Joan C. 
Published: February 2005

AbstractA pre‐embedding immunocytochemical technique is described for combined light and electron microscope study of peptidergic neurons in the central nervous system. The protocol is especially designed to overcome the sampling problems inherent in electron microscope study of structures, such as luteinizing hormone‐releasing hormone (LHRH) neurons, that are scattered individually across large brain regions. The fixation methods outlined for several mammalian species include immersion and vascular perfusion with acrolein. Fine‐structural preservation and LHRH immunoreactivity obtained with this fixative are compared to results with more conventional fixatives. Vibratome sectioning and a “pretreatment” regime, which prepare the tissues for immunocytochemistry, are described. Immunocytochemical labeling is done with free‐floating sections and the peroxidase‐antiperoxidase unlabeled antibody enzyme technique. Techniques are also described for the subsequent processing of immunoreacted sections for electron microscopy. These methods ensure that the processed sections are readily scanned by light microscopy, so that regions containing immunoreactive structures can be specifically chosen for electron microscope analysis. Sample electron micrographs are shown that illustrate some fine structural features of LHRH neurons in rats, bats, ferrets, and monkeys, as revealed with the techniques described.
Ultrastructural characteristics of peptidergic neurons using pre‐embedding immunocytochemical methods
Authors: Piekut Diane T. 
Published: February 2005

AbstractThe immunocytochemical staining technique represents a valuable method for electron microscopic analysis with which to elucidate the ultrastructural characteristics, synaptology, and potential interactions of selectively labeled neuronal systems. The unlabeled antibody peroxidase‐antiperoxidase (PAP) technique is one of the most widely used immunocytochemical methods and has been applied to both pre‐embedding and postembedding EM procedures. This review provides a detailed protocol for the former method and discusses advantages and disadvantages associated with the use of both approaches. A descriptive fine structural analysis of peptidergic neurons localized in the hypothalamus of brain illustrates the applicability and specificity of utilizing this method for the localization of cellular antigens of other systems.
Some improvement in tissue preparation and colloidal‐gold immunolabeling for electron microscopy
Authors: Hisano S. , Adachi T. , Maegawa M. , Daikoku S. 
Published: February 2005

AbstractThe application of freeze‐substitution (FS) and freeze‐drying (FD) techniques and the protein A‐gold‐antibody complex immunocytochemical methods are described. The two tissue‐preparation techniques produced excellent ultrastructure and topographical fixation of antigens when compared with conventional tissue‐preparation techniques. In the FS preparation, however, occasional extragranular immunolabeling was recognized. This may suggest the leakage of antigens from the secretory granules. The FD procedure was considered the best, since such labeling was almost negligible. The protein A‐gold‐antibody complexes are easily prepared and label the antigens clearly. If the protein A–coated gold particles are saturated with antibodies, there is no interaction between gold particles. Thus, multiple antigens can be determined even in single secretory granules. In fact, we demonstrated intragranular colocalization of immunoreactive oxytocin, labeled with 50‐nm gold particles, and immunoreactive methionine‐enkephalin, labeled with 15‐nm gold particles, in the axonal terminals of the FD‐prepared rat neurohypophysis. This study demonstrates the value of the use of gold‐antibody complexes for immunocytochemical labeling on FS‐ or FD‐treated tissues.
Postembedding immunogold labeling for electron microscopy using “LR White” resin
Authors: Timms B. G. 
Published: February 2005

AbstractA method is described for performing postembedding immunogold immunocytochemistry on sections of LR White‐embedded tissues. Fixation of tissue in a combination of paraformaldehyde and glutaraldehyde, or with low concentrations of glutaraldehyde followed by partial dehydration, resulted in preservation of antigenicity for a variety of proteins in different tissue samples. Good structural preservation facilitated high‐resolution immunolabeling when coupled with the use of purified monoclonal antibodies. The technique is straightforward and versatile, offering the potential for many immunocytochemical applications with minimal modifications.
Use of immunoperoxidase and immunogold methods in studying prolactin secretion and application of immunogold labelling for pituitary hormones and neuropeptides
Authors: Papka R. E. , Yu S. M. , Nikitovitch‐Winer M. B. 
Published: February 2005

AbstractTwo immunocytochemical methods, immunoperoxidase and immunogold (IG), were used in an attempt to study the dynamic process of prolactin release from stimulated rat pituitary mammotrophs. The immunogold method was also used to localize other pituitary hormones including growth hormone, follicle‐stimulating hormone, luteinizing hormone, and the neuropeptides substance P, neuropeptide tyrosine, leu‐enkephalin, and atrial natriuretic factor in peripheral nerves. Light‐microscopic immunoperoxidase staining of prolactin revealed a unique distribution of immunoreactive mammotrophs. Two groups of cells were seen, one centrally located and one forming a narrow peripheral rim on the gland. The two groups were separated by a zone of nonimmunoreactive cells. In addition, the distribution of immunoper‐oxidase‐stained material was not uniform in all mammotrophs. In some, prolactin immunoreactive material was clumped near the nucleus (in the Golgi cisternae); in others it was more diffused within the cytoplasm (but immediately surrounding the cisternae of rough endoplasmic reticulum). After stimulation of mammotrophs, via suckling, prolactin‐immunoreactive material was visualized in extracellular spaces. With immunogold methods, prolactin labelling was seen mainly in secretory granules; but some labelling of Golgi cisternae and rough endoplasmic reticulum also occurred. Immunogold labelling revealed that material immunoreactive for leu‐enkephalin and atrial natriuretic factor was present in nerve terminals in the rat paracervical ganglion. Material immunoreactive for substance P and neuropeptide tyrosine was present in nerve terminals in the guinea pig heart. Thus, in some situations the immunoperoxidase technique was useful and helped to visualize “grossly” the presence of specific antigens, but it was inadequate for fine ultrastructural localization of these antigens. The immunogold technique was excellent for precise localization of antigens and especially for the detection of colocalization of different antigens. This method can be used in very different structures, such as the adenohypophysis and peripheral nervous tissue, without any modification except for the nature of the antibodies.
Pitfalls in immunocytochemistry with special reference to the specificity problems in the localization of neuropeptides
Authors: van Leeuwen Fred 
Published: February 2005

AbstractIn this review, the different types of specificity in immunocytochemistry (ICC) are discussed. Some examples of misinterpretations on the basis of ICC results are given. A strategy is proposed to assess the specificity of antisera and to test them again after purification.
A comparative study of muscle spindles in slow and fast neonatal muscles of normal and dystrophic mice
Authors: Johnson Marjorie I. , Ovalle William K. 
Published: February 2005

AbstractMuscle spindles from the slow‐twitch soleus and the fast‐twitch extensor digitorum longus (EDL) muscles of genetically dystrophic mice of the dy2J/dy2J strain were compared with age‐matched normal animals at neonatal ages of 1–3 weeks according to histochemical, quantitative, and ultrastructural parameters. Intrafusal fibers in both the soleus and EDL exhibited similar regional differences in myosin ATPase activity, and conformed to those noted previously in various adult species. In distal polar regions, all nuclear bag fibers resembled extrafusal fibers of the type 1 variety, whereas in capsular zones they could be divided into two subtypes. Nuclear chain fibers possessed a staining pattern similar to type 2 extrafusal fibers, and in contrast to the bag fibers they exhibited no regional variations. These features were consistently observed in both the normal and dystrophic muscles at all ages. Spindles varied only slightly in their number and distribution in the two types of muscle, and their location followed the neurovascular branching pattern in each. Irrespective of age or genotype, spindles in the soleus were more homogeneously dispersed, but those in the EDL were concentrated along the dorsal aspect of the muscle. No significant differences were noted in the total number of spindles between normal and dystrophic muscles. In addition, no dramatic differences were observed in the muscle spindle index for soleus and EDL. The first obvious disease‐related changes were noted in extrafusal fibers of the soleus of 3‐week‐old mice, and spindles were often located close to these areas of fiber degeneration. Despite alterations in the surrounding tissue, however, spindles appeared morphologically unaltered in dystrophy. These observations indicate that intrafusal fibers of spindles in neonatal mice appear enzymatically and histologically unaffected in incipient stages of progressive muscular dystrophy.
SEM observations of the elastic networks in canine femoral artery
Authors: Crissman Robert S. 
Published: February 2005

AbstractScanning electron microscopy was used to study the normal architectural arrangement of elastic tissue in a medium‐sized muscular artery. Selective NaOH sonication digestion or formic acid digestion was used to expose and isolate the elastic networks in the femoral arteries of four healthy dogs. The digested segments were neutralized and freeze‐dried before mounting for scanning electron microscopy (SEM) observation. The fenestrated internal elastic lamina (IEL) had a smooth surface with scattered regions of the fine elastic fibers that made up lacy networks protruding from the luminal surface. Prominent ellipsoid fenestrae, randomly scattered across the surface, were grouped into small and large sizes based on their mean diameter. The openings of most fenestrae were bridged by elastic fibers to give the fenestrae a sieve‐like appearance. Large, transversely oriented, fusiform gaps were randomly scattered along the length of the IEL. These gaps, filled in by an elastic fiber network, sometimes spanned as much as a quarter of the vessel circumference. It is suggested that these gaps represent splits in the IEL that have been repaired. The tunica media contained a complex network of anastomosing elastic fibers and lamellae that were primarily circumferential in orientation. A well‐defined external elastic lamina formed a solid sheet at the junction of the tunica media and the tunica adventitia. The tunica adventitia contained 8–10 incomplete lamellae of large, interconnecting, longitudinally oriented fibers. The architecture of the elastic network in canine femoral artery was compared with that previously described in medium‐sized canine veins and in the rat femoral artery.
Tooth induction and temporal patterning in palatal epithelium of fetal mice
Authors: Richman Joy Marion , Kollar Edward J. 
Published: February 2005

AbstractThe present study examined the effect of aging on epithelium and on its ability to respond to an inductive stimulus provided by murine dental papillae. In fetal CD‐1 mice, 15‐ to 17‐day molar mesenchyme was combined with 15‐ to 19‐day epithelium from the secondary palates. Enamel organs were separated from the dental papillae, and palatal epithelium was peeled away from its underlying mesenchyme after treatment with 1% trypsin. Recombinants of epithelium and papillae were initially cultured on a solidified complex medium for 24 hr followed by an additional 10–14 days of intraocular explanation. Control specimens consisted of isolated molar papillae. Nineteen of 88 isochronal, heterotypic recombinations formed teeth. None of the 46 heterochronal, heterotypic grafts of 18‐ and 19‐day palatal epithelium combined with 15‐ to 17‐day molar papillae‐produced teeth. Instead, keratin‐filled epithelial cysts and bone spicules were formed. Isolated control molar papillae often formed bone in the intraocular sites but did not form teeth or contain epithelium. These results show that palatal epithelium is first restricted to its developmental pathway at 18 days of gestation. Younger epithelium can convert to functional ameloblasts that secrete enamel protein. In addition to the change in gene expression, normal tooth morphology is attained. The loss of competence of the palatal epithelium at 18 days gestation coincided with the acquisition of stratum corneum and the attainment of the fully differentiated state. The oral surface of palatal epithelium appears to be determined histogenically and morphogenically at 18 days of gestation in mice.
Collagen types I, III, and V in human embryonic and fetal skin
Authors: Smith Lynne T. , Holbrook Karen A. , Madri Joseph A. 
Published: February 2005

AbstractThe dermis of human skin develops embryonically from lateral plate mesoderm and is established in an adult‐like pattern by the end of the first trimester of gestation. In this study the structure, biochemistry, and immunocytochemistry of collagenous matrix in embryonic and fetal dermis during the period of 5 to 26 weeks of gestation was investigated. The dermis at five weeks contains fine, individual collagen fibrils draped over the surfaces of mesenchymal cells. With increasing age, collagen matrix increases in abundance in the extracellular space. The size of fibril diameters increases, and greater numbers of fibrils associate into fiber bundles. By 15 weeks, papillary and reticular regions are recognized. Larger‐diameter fibrils, larger fibers, denser accumulations of collagen, and fewer cells distinguish the deeper reticular region from the finer, more cellular papillary region located beneath the epidermis. The distribution of collagen types I, III, and V were studied at the light microscope level by immunoperoxidase staining and at the ultrastructural level by transmission (TEM) and scanning electron microscopy (SEM) with immunogold labeling. By immunoperoxidase, types I and III were found to be evenly distributed, regardless of fetal age, throughout the dermal and subdermal connective tissue with an intensification of staining at the dermalepidermal junction (DEJ). Staining for types III and V collagen was concentrated around blood vessels. Type V collagen was also localized in basal and periderm cells of the epidermis. By immuno‐SEM, types I and III were found associated with collagen fibrils, and type V was localized to dermal cell surfaces and to a more limited extent with fibrils. The results of biochemical analyses for relative amounts of types I, III, and V collagen in fetal skin extracts were consistent with immunoperoxidase data. Type I collagen was 70–75%, type III collagen was 18–21%, and type V was 6–8% of the total of these collagens at all gestational ages tested, compared to 85–90% type I, 8–11% type III, and 2–4% type V in adult skin. The enrichment of both types III and V collagen in fetal skin may reflect in part the proportion of vessel‐ and nerve‐associated collagen versus dermal fibrillar collagen. The accumulation of dermal fibrillar collagen with increasing age would enhance the estimated proportion of type I collagen, even though the ratios of type III to I in dermal collagen fibrils may be similar at all ages.