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PubMed1961

Observations on the fine structure and cytochemistry of mouse and human intercostal neuromuscular junctions.

The fine structure of the mouse and human intercostal muscle neuromuscular junction was studied after brief fixation in a new formol-sucrose fixative. This primary formalin fixation was followed by brief postosmication in buffered 1 per cent osmium tetroxide. Muscle blocks were embedded in methacrylate or Epon 812 epoxy resin. Marked similarities between mouse and human motor end-plates were observed. Neuromuscular junctions from both mouse and human intercostal muscle showed synaptic vesicles, primary and secondary synaptic clefts, and layered differentiation of the amorphous surface material (ASM) present on the surface of the Schwann cell plasma membrane and on the muscle surface membrane in the region of the neuromuscular junction. An attempt to stain the ASM with lead was unsuccessful. Observations on thick and thin plastic-embedded sections stained by PAS after diastase digestion showed that the ASM within the subneural apparatus is PAS positive. Alcian blue stained the endoneurium and perineurium of peripheral nerve bundles and portions of the end-plates. The similarity of the PAS-positive ASM to other basement membranes described in other sites is discussed and its possible physiologic significance within the subsynaptic apparatus is considered.

Animals↗
PubMed1999

Development of the chick olfactory nerve.

Gonadotropin releasing hormone (GnRH) is produced and secreted by neurons dispersed throughout the septal-preoptic and anterior hypothalamic areas in adult birds and mammals. These neurons, essential for a functional brain-pituitary-gonadal axis, differentiate in the olfactory placode, the superior aspect of which forms the olfactory epithelium. To reach their final placement within the brain, GnRH neurons migrate out of the epithelium and along the olfactory nerve to the CNS. This nerve is essential for the entrance of GnRH neurons into the CNS. Due to the importance of the nerve for the proper migration of these neurons, we have used immunocytochemistry, DiI labeling and 1 microm serial plastic-embedded sections to characterize the nerve's earliest development in the embryonic chick (stages 17-21). Initially (stage 17) the zone between the placode and prosencephalon is a cellular mass contiguous with the placode. This cluster, known as epithelioid cells, is positive for some but not all neuronal markers studied. The epithelium itself is negative for all neuronal and glial markers at this early stage. By stage 18, the first neurites emerge from the epithelium; this was confirmed at stage 19 by examination of serial 1 microm plastic sections. There is sequential acquisition of immunoreactivity to neuronal markers from stage 18 to 21. The glial component of the nerve appears at stage 21. Axons originating from epithelium, extend to the border of the CNS as confirmed by DiI labeling at stage 21. Small fascicles have entered the CNS at this stage. As previously reported, GnRH neurons begin their migration between stages 20-21 and have also arrived at the border of the brain at stage 21. Despite the penetration of neurites from the olfactory nerve into the CNS, GnRH neurons pause at the nerve-brain junction until stage 29 (2 1/2 days later) before entering the brain. Subsequent studies will examine the nature of the impediment to continued GnRH neuronal migration.

Acetylcholinesterase↗
PubMed1998

Peripherin-like immunoreactivity in type II spiral ganglion cell body and projections.

Peripherin, an intermediate filament protein, is present in neuronal subpopulations of both peripheral and central nervous systems. The distribution of peripherin was studied in the adult rat cochlea using immunohistochemistry on whole mount material, in cryostat sections and sections of plastic embedded tissue. In the spiral ganglion, peripherin labeling was restricted to the perikarya of a subpopulation of neurons and their peripheral and central processes. Peripherin positive neurons had the following features: (i) they have a large eccentric nucleus, they were often found in a cluster of 2 or 3 cells, (ii) they were often located near the intraganglionic spiral bundle fibers, (iii) they represented roughly 8% of the whole ganglion population and (iv) on the average they had smaller perikarya than non-immunoreactive cells. Immunostaining on semithin plastic sections revealed positive reactivity on Type II ganglion cells, while Type I neurons were negative. Double labeling using peripherin and three neurofilament (NF) subunit antibodies confirmed the presence of both markers within the same spiral ganglion cell type. Type II neurons have been previously documented as the only subpopulation of the spiral ganglion that presents a strong positive NF immunoreactivity within their perikarya. In the organ of Corti, peripherin-positive fibers formed bundles that course beneath the outer hair cells and send branches that end as boutons contacting the outer hair cells. All these characteristics suggest that peripherin-positive cells are Type II neurons, and that peripherin constitutes a reliable marker for this spiral ganglion subpopulation, as well as their peripheral and central processes.

Animals↗
PubMed1975

Light microscopic differential staining of epoxy-embedded adenohypophysis.

Light microscopic histochemical methods for differential staining of adenohypophysis glandular cells were applied to Epon-embedded tissue, after prior softening of resin with saturated solution of NaOH in absolute ethanol. All investigated staining procedures, i.e. carmoisin L-orange G, PAS-orange G, PAS-range G-methyl blue, Gabe's aldehyde fuchsin-Halmi counterstain, aldehyde thionin-PAS-orange G and performic acid-alcian blue-PAS-orange G were found to give satisfactory results and tissue structure showed no signs of damage caused by the Epon-dissolving reagent. The features of light microscopic image given by plastic-embedded semithin sections: nearly 2-dimensional picture as well as the highest possible resolving power provide the opportunity for much more precise and detailed examination of adenohypophysis prepared that way, as compared with conventionally paraffin-embedded gland. Moreover, the described technique permits to establish a direct connection between light microscopic differential histochemistry of adenohypophysis and the electron microscopy by means of adjacent sections.

Animals↗
PubMed1989

Histochemical identification of the vascular endothelial isoenzyme of alkaline phosphatase.

Alkaline phosphatase (AP) is a widely studied membrane bound ecto-enzyme with an extensive distribution in nature. Three major human isoenzymes have been defined and can be distinguished on the basis of their differential sensitivity to specific inhibitors. Despite the voluminous literature describing AP, the physiological role of this enzyme is unclear. Microvascular endothelium is strongly AP positive and may provide a convenient model for study of the role of AP in vitro. This report describes the use of freeze-substitution and high-resolution plastic embedding techniques to identify the isoenzyme of endothelial AP by quantitative analysis of the relative inhibition by specific inhibitors of AP, using human gingival tissues and a number of rat tissues. Endothelial AP is found to be the liver/bone/kidney isoenzyme, indicating kidney as a credible source of enzyme for further experimental work investigating the role of AP.

Alkaline Phosphatase↗
PubMed1980

High resolution optical microscopy of animal tissues by the use of sub-micrometer thick sections and a new stain.

The introduction of 1 micron-thick sections from plastic embedded material represents a great technical improvement for the study of tissues under the optical microscope. However, even sections of this thickness appear too thick when observed with oil immersion objectives of high numerical aperture. The extremely shallow depth of field of these lenses allows them to differentiate several focal planes within a one micron thick section. This in turn results in ghost images being formed from out of focus structures, a problem particularly vexing when photomicrography is attempted. To circumvent this difficulty, we reduced the thickness of the sections down to an optimum of 0.4 micron. These thinner sections do require a very energetic stain to give enough contrast to the cellular structures; Stevenel Blue, a stain recently adapted for plastic sections [del Cerro et al., Microsc. Acta 83, (2), 117--121 (1980)] proved to be the most suitable for this purpose of several stains tested. In summary, submicrometer thick sections stained with Stevenel Blue allow to reach the limits of visibility permitted by the best available objectives and effectively merge the realm of optical microscopy with that of low power electron microscopy.

Animals↗
PubMed2006

Heat-induced antigen retrieval: mechanisms and application to histochemistry.

Since the introduction of the fluorescence-labeled antibody method by Coons et al. [Immunological properties of antibody containing a fluorescent group. Proc Soc Exp Biol Med 47, 200-2002], many immunohistochemical methods have been refined to obtain high sensitivity with low background staining at both light and electron microscopic levels. Heat-induced antigen retrieval (HIAR) reported by Shi et al. in the early 1990s has greatly contributed to immunohistochemical analysis for formalin-fixed and paraffin-embedded (FFPE) materials, particularly in the field of pathology. Although antigen retrieval techniques including enzyme digestion, treatment with protein denaturants and heating have been considered tricky and mysterious techniques, the mechanisms of HIAR have been rapidly elucidated. Heating cleaves crosslinks (methylene bridges) and add methylol groups in formaldehyde-fixed proteins and nucleic acids and extends polypeptides to unmask epitopes hidden in the inner portion of antigens or covered by adjacent macromolecules. In buffers having an appropriate pH and ion concentration, epitopes are exposed without entangling the extended polypeptides during cooling process, since polypeptides may strike a balance between hydrophobic attraction force and electrostatic repulsion force. Recent studies have demonstrated that HIAR is applicable for immunohistochemistry with various kinds of specimens, i.e., FFPE materials, frozen sections, plastic-embedded specimens, and physically fixed tissues at both the light- and electron-microscopic levels, and have suggested that the mechanism of HIAR is common to aldehyde-fixed and aldehyde-unfixed materials. Furthermore, heating has been shown to be effective for flow cytometry, nucleic acid histochemistry (fluorescein in situ hybridization (FISH), in situ hybridization (ISH), and terminal deoxynucleotidyl transferase-mediated nick labeling (TUNEL)), and extraction and analysis of macromolecules in both FFPE archive materials and specimens processed by other procedures. In this article, we review mechanism of HIAR and application of heating in both immunohistochemistry and other histochemical reactions.

Animals↗
PubMed1993

Scanning electron microscopic distinction of pleural mesotheliomas from adenocarcinomas.

Cases of mesothelioma and adenocarcinoma involving pleura were examined by light and scanning electron microscopy utilizing H & E stained slides of paraffin-embedded material. Areas of interest were identified and isolated, and the coverslips were removed. The slide fragments were mounted on scanning electron microscopy stubs and coated with gold for 45 sec. Cases were examined in a blinded manner. Microvilli were readily identified by scanning electron microscopy with preservation of morphologic detail through the processing and staining steps required for light microscopy. The length and diameter of 20 microvilli were measured from each tumor. The mean length:diameter ratio for the mesotheliomas (n = 7) was 19.7:1 (range 13.7-23.5:1), and for the adenocarcinomas (n = 7), 2.5:1 (range 1.3-4.1) (P < 0.0005). Correlative transmission electron microscopy was performed on "scanned" specimens, and length:diameter ratios were generated. This technique has utility in distinguishing mesothelioma from adenocarcinoma. It is especially useful when glutaraldehyde-fixed, plastic-embedded tissue is not available for transmission electron microscopy. Additionally, scanning electron microscopy allows more extensive examination than transmission electron microscopy and is far less limited by section thickness and tangential sectioning artifact.

Adenocarcinoma↗
PubMed1975

Ice crystal growth in skeletal muscle fibres.

Ice crystal growth was studied in rapidly frozen skeletal muscle fibres which were treated with cryo-protective additives (glycerol, DMSO, sucrose) or which were untreated. Freeze cleaving and etching was the basic method, with conventional plastic embedding and cryo-ultramicrotomy arred during freezing in all unprotected fibres. Just below the fibre surface the crystals were numerous but small, while deeper in the fibre they were fewer but larger. The deeper within the specimen a fibre was located, the larger, in general, was the crystal size. The crystal volume density was about 55%, irrespective of crystal size. Ice recrystallization was practically absent at the temperature normally used in cryo-sectioning (-70 degrees C). Anti-freeze treatment eliminated crystal growth. If the anti-freeze agents were used in non-toxic concentrations, however, their effect on crystal growth was very limited. 'Dry'-cut, freeze-dried ultra-thin cryosections of protected and unprotected fibres confirmed these observations, while sections obtained by 'wet' cryo-cutting showed no apparent signs of crystal growth. In plastic sections of frozen and thawed fibres a previous occurrence of crystals was only slightly indicated. In interpreting the ultrastructure in 'wet'-cut cryo-sections of unprotected frozen muscle fibres, the distorting effects of ice crystals through mechanical compression and alterations in sectioning conditions, must be taken into consideration. Crystal growth also strongly limits the possibilities of using 'dry'-cut sections of untreated frozen tissue for analytical electron microscopy; only the most superficial parts of the fibres seem to be suitable for microanalysis.

Animals↗
PubMed1987

Histomorphometric identification of carbonic anhydrase in fetal rat bone embedded in glycolmethacrylate.

Carbonic anhydrase was identified in bone-resorbing cells present in sections of fetal rat femur embedded in glycolmethacrylate. Using a slight modification of the Hansson's histochemical method, we demonstrated that most chondroclasts (91.8-95.4%) and osteoclasts (95.1-96.3%) display a positive histochemical reaction for carbonic anhydrase. This staining was consistently inhibited in the presence of very low concentrations (10(-6), 10(-7) M) of the specific inhibitor acetazolamide. The number of chondroclasts reacting for carbonic anhydrase was identical to the number of acid phosphatase-stained chondroclasts determined on adjacent sections. A large majority of osteoclasts (96.3%) stained for carbonic anhydrase and for acid phosphatase (97.2%), with more osteoclasts reacting for the latter enzyme than the former (76.8 +/- 8.5 (SD) vs 85.3 +/- 9.2 cells/mm2 of endosteal bone; p less than 0.01). The observation that acetazolamide at a concentration as low as 10(-7) M inhibited Hansson's reaction, together with our histomorphometric results, validates the use of histochemical staining for carbonic anhydrase to evaluate activity of bone-resorbing cells identified in plastic-embedded fetal bone tissue.

Acrylates↗
PubMed1990

Scanning ion microprobe assessment of biological sample preparation techniques.

Different preparation techniques for high lateral resolution scanning ion microprobe imaging of biological samples have been investigated. The sharpest histological maps are obtained from chemically fixed and plastic embedded specimens. It is often problematic to correlate ultrastructure and bioaccumulation from analysis of frozen cut and lyophilized sections. The best compromise is to resin-embed frozen samples in order to get a perfectly flat section from tissue where the in vivo ion distribution is maintained. Use of the University of Chicago Ion Microprobe gave us the ability to observe the relative ion translocations induced during sample preparation. As an example, we show the rapid decrease of intracellular K+/Na+ ratio through a fast frozen blood droplet.

Blood Cells↗
PubMed1983

New methods for cathodoluminescence in the scanning electron microscope.

Experiments using the CL imaging mode to recognise osteoid in the polished, cut surfaces of bone biopsies embedded in PMMA led to the development of a number of new methods for contrast formation in CL images in the SEM. These involve: (1) enhancing or (2) reducing the CL signal by staining the specimen, (3) utilising the cathodoluminescence of glass microscope slides to produce images of histological sections mounted on glass so that features in the section which scatter the electron beam appear dark against a light background, and (4) enhancing the CL signal from PMMA so that features which are less penetrated by the scintillator show up dark against a bright background. Efforts to increase the efficiency of light collection resulted in the development of a new means for manufacturing reflector-cum-light guide CL detectors by wrapping aluminum foil around a wooden former. These detectors enshroud the specimen so that CL light can only escape to the photomultiplier window (or back up the final lens). A variety of such designs have proved more efficient than the conventional plastic light guides used as CL detectors. By enlarging the beam entry aperture, other SE and BSE detectors can be used simultaneously. Examples of the value of the CL mode in mineralised tissue research include the use of enhanced CL plastic embedding media to detect marrow space and of enhanced osteoid CL to detect unmineralised bone matrix; the use of tetracycline as a growth marker in pathological studies of bone and experimental studies with bone, dentine and enamel; the use of bisbenzamid to locate and count nuclei in osteoclasts, the hard tissue resorbtive cells; and the use of superficial stain absorption of auto-CL to locate stained material on tooth surfaces, with the view to monitor the efficiency of periodontal therapy.

Animals↗
PubMedDate not supplied

A further evaluation of the effect of age on striate cortex of the rhesus monkey.

The brains of 14 rhesus monkeys (Macaca mulatta) between 4 and 35 years old were examined to determine the effects of aging on the thickness, neuronal frequency, fine structure, surface area, and volume of striate cortex. The effects of aging were ascertained by comparing the striate cortex in the six monkeys between 4 and 12 years of age with that of the eight monkeys over 25 years of age. The brains of the monkeys were all fixed by vascular perfusion and except for one of the old monkeys, whose age was estimated, the exact ages of all of the monkeys are known. One micron thick sections of plastic embedded cortex from one hemisphere of each monkey were examined by light microscopy to determine the thickness of the striate cortex, and neuronal frequency was determined by counting the numbers of neurons displaying nuclei in 250 microns-wide strips passing through the thickness of the cortex. When young monkeys were compared with the old ones, no differences were found in either the thickness of the cortex or in the numbers of neuronal profiles beneath unit areas of cortical surface. This suggests that neurons are not lost with age, and when the cortices were examined by electron microscopy there was no indication that the cell bodies of neurons are degenerating, except possibly in layer 1. Serial, 30 microns-thick, Nissl stained frozen sections from the other hemisphere of each monkey were used to determine both the surface area and the volume of the striate cortex. Overall, the surface area varied between 702 and 1480 mm2, with a mean value of 956 mm2, but there was no indication that the surface area decreased with age, and the same is true for the volume of striate cortex. The conclusion is that while there is a large variation in the amount of cortex occupied by area 17, there is no indication that its thickness, volume, or number of neurons is altered by age.

Aging↗
PubMed1981

Development of myelin in human optic nerve and tract. A light and electron microscopic study.

The early development of myelin in human optic nerve and tract was studied in plastic-embedded material from post-mortem examinations of 18 infants and children. Specimens were examined by light and electron microscopy, and a good correlation was found between the data obtained by the two techniques. The characteristic lamellae of myelin were first seen around a few fibers of tract and intracranial optic nerve at 32 weeks of gestation. By term, these myelin sheaths had become thicker, and a majority of the nerve fibers had become myelinated. In the optic nerve near the globe, myelin was first seen at term and virtually all fibers were myelinated by 7 months of age. Significant increases in sheath thickness were seen in the first two years, and modest increases were found thereafter. These results are in agreement with earlier observations that, in the optic nerve, myelination proceeds from the brain toward the eye. The present data suggest that a significant amount of myelination in the human optic nerve occurs after a full-term (40-week) gestation, during a period of rapid postnatal visual development.

Adolescent↗
PubMed1984

Corneal nerve alterations in diabetes mellitus.

The morphologic status of corneal innervation was studied in rats with streptozocin-induced diabetes. Animals were killed at 1, 4, 16, and 36 weeks. Corneal innervation was studied by light and electron microscopy using nonspecific cholinesterase reaction, gold chloride impregnation, and plastic-embedded sections. Increased irregularity in the periodicity of nerve fiber beading was observed in diabetic corneas with gold impregnation. Ultrastructural evidence of irregularities in the basal lamina of Schwann cells was demonstrated in 16- and 36-week-old diabetic animals, along with occasional axonal degeneration. These alterations constitute a constellation of early pathologic manifestations in the innervation of diabetic cornea. To our knowledge, this study represents the first demonstration of neural changes in diabetic corneas as well as nerve fiber changes in an avascular tissue in diabetes.

Animals↗
PubMed1987

Müller's cell involvement in proliferative diabetic retinopathy.

To test whether the Müller's cell, and not the fibrous astrocyte alone, is an active glial element in proliferative diabetic retinopathy, we developed a method using enzyme histochemical analysis for carbonic anhydrase and immunolabelling for glial fibrillary acidic protein on plastic-embedded sections to detect Müller's cells in degenerated retinas. Four postmortem eyes with proliferative diabetic retinopathy were studied. Four features of Müller's cell behavior were observed: (1) Müller's cell nuclei migrated within the retina. (2) They were the principal glial cells in preretinal membranes. (3) Müller's cells formed horizontal bands, in the outer retina, that gave the appearance of subretinal membranes. (4) They formed intraretinal bridges between cystic spaces. In conclusion, the Müller's cell (or its progeny) plays an active role in proliferative diabetic retinopathy.

Adult↗
PubMed1992

Protective effects of flunarizine on ischemic injury in the rat retina.

Intracellular calcium overload has been implicated to be a major factor in triggering cell death after ischemic neuronal injury. We investigated the effects of flunarizine hydrochloride, a calcium-overload blocker, on pressure-induced retinal ischemia in a rat model. Retinal ischemia was induced in intraocular pressure to 110 mm Hg for 45 minutes. Two regimens of treatment with flunarizine were examined: (1) prophylactic treatment, in which flunarizine was administered before ischemia and in the early phase of reperfusion; and (2) postischemic treatment, in which flunarizine was administered only in the early phase of reperfusion. Injury was evaluated morphologically and morphometrically by measuring the thickness of the inner retinal layers on plastic-embedded retinal sections and by counting the retinal ganglion cells on retinal flat preparations. By morphologic and morphometric criteria, a significant but partial protection of the inner retinal layers was noted in the groups given either regimen. This protective effect of flunarizine suggests that elevated intracellular calcium concentration may play an important role in ischemic retinal injury.

Animals↗
PubMed1998

Germ cell kinetics during early ovarian differentiation: an analysis of the oogonial cell cycle and the subsequent changes in oocyte development during the onset of meiosis in the rat.

The aim of this study was the comparison between the mitoses of oogonia and the initial stages of oocyte meiosis. The structural alterations that the germ cell chromatin undergoes during the oogonial mitosis have been compared with those occurring during the G1- and S-phase just before meiosis. Using plastic embedded 1-microm sections of fetal rat ovaries (embryonic days = ED 14-20) labeled with 3H-thymidine and re-embedded for electron microscopy, a study of the structural conditions of the nuclear chromatin has been combined with a kinetic analysis of the oogonial cell cycle and the transitional period into the meiotic prophase. After ovarian differentiation (ED 14) the oogonia show a non-clonal, but strong proliferation. On ED 16, proliferation changes to a clonal pattern and decreases during ED 17. A final increase in 3H-thymidine incorporation on ED 18 characterizes the meiotic S-phase. On ED 19 the nuclear labeling drops to zero. The mitotic cycle of the oogonia lasts 16.5 hr and can be divided into 11 stages according to the concept of El-Alfy and Leblond [(1988) Am. J. Anat., 183:45-56] on the basis of the chromatin pattern. The S-phase (10.0 hours) extends from the telophase-interphase transition through the interphase to early prophase. The postmitotic G1- and S-phases show a more extensive duration, respectively 10 and 11.5 hours, and differ from their oogonial counterparts by the spherical shape of the nuclei from the very beginning. The chromatin pattern is similar until the end of the S-phase and lacks any prophase-like, preleptotenal chromatin condensation before the oocytes exhibit (pre-) leptotenal structures. Once the germ cell has completed a sequence of clonal mitotic divisions, it irrevocably progresses into meiosis. During an extended postmitotic period, the structural characteristics of meiosis emerge stepwise.

Animals↗