bluuebunny/crossref_metadata_2025 huggingface dataset:
Tracing Central Pathways with the Autoradiographic Method,
Authors: Swanson L.W.
Published: April 2011

The major advantages and disadvantages of the autoradiographic method for tracing pathways in the central nervous system have become clear in the decade since its introduction. Attention is focused here on two major problems associated with the interpretation of autoradiographic experiments. First, it is often difficult to determine the effective size of an injection site, which may be different for different projections in the same experiment. Quantitative evidence presented here also shows that the apparent size of injection sites in the hippocampus decreases considerably in diameter 1 to 2 weeks after the injections are made. And second, the morphology of labeled pathways must be inferred from a pattern of silver grains lying over the tissue section. Several examples are used to illustrate the point that the autoradiographic method should be used in conjunction with retrograde transport, immunohistochemical, and electron microscopic techniques to investigate the origin, course, and synaptic relationships of individual pathways in the brain and spinal cord.
Quantitative Analysis of Steroid Autoradiograms,
Authors: Arnold Arthur P.
Published: April 2011

Two types of quantitative criteria are compared that have been used to discriminate labeled and nonlabeled cells in steroid autoradiograms: multiplicative criteria and a criterion based on the Poisson distribution. Several factors affect the accuracy of the assessment of cell labeling, including variations in the length of exposure, variations in cell size, and possible nonlinearities in the grain density of autoradiograms as a function of exposure time. These factors have a greater effect on the accuracy of the determination of cell labeling when using multiplicative criteria, so that the Poisson criterion is more suitable.
Concurrent acetylcholinesterase staining and gamma-aminobutyric acid uptake of cortical neurons in culture.
Authors: Mesulam M M , Dichter M
Published: April 2011

Gamma-aminobutyric acid (GABA) uptake and acetylcholinesterase (AChE) content were demonstrated concurrently in cortical neurons grown in tissue culture. Positive reactions either for GABA uptake or for AChE content were encountered in pyramidal and stellate, as well as spindle-shaped neurons. Neither reaction was confined to a specific morphological subtype. Nearly half the neurons were negative for either reaction. Most of the remaining neurons were positive only for GABA or only for AChE. However, a subpopulation of neurons showed not only a high AChE content, but also an avid GABA uptake. Thus, four types of neurons could be identified on the basis of these two reactions. The high AChE content in some of the cortical neurons that also showed GABA uptake indicates that there are at least two distinct types of GABAergic neurons.
Ultrastructural demonstration of noradrenergic synapses in the rat central nervous system by dopamine-beta-hydroxylase immunocytochemistry.
Authors: Olschowka J A , Molliver M E , Grzanna R , Rice F L , Coyle J T
Published: April 2011

Noradrenergic (NA) cell bodies and axonal processes were identified in the electron microscope by the immunocytochemical localization of the norepinephrine-synthesizing enzyme, dopamine-beta-hydroxylase (DBH). DBH immunoreactivity, visualized by the peroxidase-antiperoxidase method, was observed in the somata and proximal processes of locus coeruleus neurons and in the distal axons of several NA terminal fields. DBH immunoreactivity is distributed throughout the cytoplasm of the NA neuron, but demonstrates a selective association with endoplasmic reticulum, Golgi apparatus, mitochondrial outer membranes, large granular vesicles, and small, round synaptic vesicles. DBH-positive axonal profiles, typically interspersed between unlabeled dendrites, form two distinct populations: a) thin, unmyelinated intervaricose segments (ca. 0.28 micron) and b) spherical varicosities (ca. 1.00 micron). No DBH-positive varicosities were observed in contact with intracerebral capillaries. In order to determine whether or not NA axons typically form synaptic contacts, a quantitative analysis of selected areas of the diencephalon, cerebellum, and limbic cortex was carried out. More than half (58%) of all DBH-positive varicosities form axodendritic synapses characterized by specialized junctional appositions. The results suggest that NA neurons typically exert their influence on other neurons through highly restricted synaptic contacts.
Immunocytochemical localization and identification of the major surfactant protein in adult rat lung.
Authors: Williams M C , Benson B J
Published: April 2011

We investigated the cellular and subcellular sites of metabolism of the 72,000 dalton protein of pulmonary surfactant in order to provide insights into mechanisms of synthesis, intracellular assembly, and intraalveolar metabolism of this phospholipid-rich secretory product. Surfactant (approximately 90% lipid, 10% protein by weight) was purified by density gradient centrifugation of material obtained by lavaging rat lungs. The purified material was used to generate an antiserum from which a specific antibody was obtained by affinity chromatography. A horseradish peroxidase-labeled Fab was used to localize the antigen in rat lung. The antibody labeled the rough endoplasmic reticulum and Golgi apparatus of type II cells only. Some multivesicular bodies in type II cells were also labeled, but whether the antigen was present in lamellar bodies was uncertain. Phagosomes of alveolar macrophages were labeled as were similar inclusions in type I cells. Using indirect immunocytochemistry we determined that the labeling of alveolar cell surfaces does not represent the presence of a continuous layer of secreted surfactant. These results suggest that only the type II cell synthesizes surfactant protein and than mainly alveolar macrophages participate in its catabolism. The initial intracellular site of the association of protein with lipid may be multivesicular bodies as suggested previously by others.
Quick-freeze fixation and freeze-drying of isolated rat pancreatic islets: application to the ultrastructural localization of inorganic phosphate in the pancreatic beta cell.
Authors: Dudek R W , Boyne A F , Freinkel N
Published: April 2011

A bounce-free mechanical quick-freeze assembly and a Coulter-Terracio freeze-dry apparatus were successfully coupled to obtain high quality ultrastructural preservation of pancreatic beta cells in a simple and dependable manner. Except for obvious shrinkage spaces, morphological relationships at the tissue, cellular, and subcellular levels were all intact. Beta cell secretory granules demonstrated a dense core surrounded by an electron lucent halo as typically described in specimens after aqueous fixation. Cell membranes and intracellular membranes demonstrated a trilaminar appearance. Golgi apparatus were well preserved. Two clearly defined populations of mitochondria were found. One group of very dark mitochondria had extremely dense matrices in which cristae were barely visible. A second group of mitochondria had light matrices with prominent cristae. The combined quick-freeze fixation and freeze-drying was applied to reevaluate the ultrastructural localization of inorganic phosphate that had been precipitated with lead in the beta cells of pancreatic islets. Accumulation of inorganic phosphate adjacent to the plasma membrane and over the nucleolus of the beta cell in nonstimulated islets was documented with better detail than heretofore possible.
Permeability of rat choriocapillaris to hemeproteins. Restriction of tracers by a fenestrated endothelium.
Authors: Pino R M , Essner E
Published: April 2011

The choriocapillaris is the fenestrated capillary bed in the choroid of the eye and is the major blood supply to the retinal pigment epithelium (RPE) and photoreceptor cells. Bruch's membrane (BM) is a multilaminated basement membrane that separates the choriocapillaris from the RPE. In a previous study (Pino RM, Essner E; Cell Tissue Res 208:21, 1980) we found that the choriocapillary endothelium restricted the egress of ferritin from the choriocapillaris. In the present study, hemeproteins were used to further establish the permeability characteristics of this capillary bed. Horseradish peroxidase (Einstein-Strokes radius (ESR), 30 A) rapidly crossed the capillary endothelium (less than 5 min) after intravenous administration and after 5 minutes filled BM and the basal infoldings of the RPE. In contrast, hemoglobin (Hg) (ESR, 32 A) and lactoperoxidase (LP) (ESR, approximately 40 A) are markedly restricted at the level of endothelial diaphragmed fenestrae, channels, and intercellular junctions. Little vesicular transport of these proteins was observed. The reaction product of the two hemeprotein activities was not demonstrable in BM for up to 30 min after injection; relatively low levels were detected after 75 min. HG and LP appear to be further restricted by BM, since their reaction products were not demonstrable between the RPE basal infoldings at this time. Catalase (ESR, 52 A) activity was not detected in BM for up to 4 hr after injection. These results indicate that the rat choriocapillary endothelium, unlike the fenestrated endothelia lining other vascular beds, substantially restricts the passage of large tracer molecules.
Lysosomal cathepsin B-like activity: mobilization in prereplicative and neoplastic epithelial cells
Authors: Pietras RJ , Szego CM , Roberts JA , Seeler BJ
Published: April 2011

Extracellular release of acid thiol proteinase activity by prereplicative and neoplastic epithelial cells was studied in serum-free, chemically defined media (CDM) in vitro. Cells isolated from urinary bladder of male bullfrogs and endometrium of ovariectomized rats each showed preferential secretion of cathepsin B-like (CB) activity within 30 min after exposure to carcinogenic nitrosamines (5 X 10(-4) M) or to mitogenic estrogen 1 X 10(-9) M), respectively. In contrast, release of such proteinase, and stimulation of cell proliferation were far less extensive in rat preputial gland cells treated with estradiol-17 beta. Striking secretion of CB was characteristic of neoplastic, but not noncancerous, epithelial cells from human ectocervix. Neoplastic cells with divergent rates of cell-to-cell aggregation were separated by a filtration method. Those cells with high rates of intercellular aggregation also exhibited higher rates of cell proliferation in CDM, as well as in soft gels, and a greater level of CB release than corresponding cancer cells with a relatively low degree of intercellular adhesion. Brief treatment of neoplastic cervical epithelial cells with liposomes containing entrapped leupeptin, a potent inhibitor of CB activity, elicited a sharp reduction in both cellular thiol proteinase activity and cell growth as compared to appropriate controls. These data indicate that mobilization of lysosomal CB activity in prereplicative and malignant cells may play a significant role in the promotion of cell proliferation.
A method of staining basal-cell plasma membranes in epidermal sheets.
Authors: Sarsfield P , Fitzgerald M J , Furey J P
Published: April 2011

An adenosine triphosphatase method was devised to stain basal epidermal cell plasma membranes in sheet preparations of humans, rat, mouse and guinea pig epidermis. The method is useful for direct observation of sizes, shapes, and numbers of basal epidermal cells.
Gastric proton pump localization. Application of triphosphatase and monophosphatase techniques.
Authors: Firth J A , Stranks G J
Published: April 2011

Potassium-dependent phosphatase activity can be demonstrated in unfixed frozen sections of mouse stomach using either adenosine triphosphate (ATP) or p-nitrophenyl phosphate (NPP) as substrate. In both cases the potassium-dependent reaction is confined to oxyntic cells, but with ATP, a strong, potassium-independent reaction occurs in the connective tissue of the lamina propria and elsewhere. In the NPP system potassium-independent reaction is very slight, and the oxyntic cell reaction shows responses to inhibitors that differentiate it from Na+, K+-ATPase and that are consistent with its identification with the dephosphorylation step of the proton pump enzyme H+, K+-ATPase, recognized as the active transport component in gastric acid secretion.
Immunohistochemical localization of dipeptidyl aminopeptidase IV in rat kidney, liver, and salivary glands.
Authors: Fukasawa K M , Fukasawa K , Sahara N , Harada M , Kondo Y , Nagatsu I
Published: April 2011

Specific antibodies directed against dipeptidyl aminopeptidase (DAP) IV prepared from rat and pig kidneys were produced in rabbits. Using a peroxidase-labeled or fluorescein isothiocyanate-labeled antibody method, localization of DAP IV was investigated in various tissues of the rat (kidney, liver and salivary glands). DAP IV was mainly localized in the brush border of the proximal tubules (kidney), in the cell membrane around bile canaliculi of hepatic cells (liver), and in the cell membrane of serous acinar cells (parotid and submaxillary glands). These results substantiate that DAP IV is a membrane-bound peptidase.
Horseradish peroxidase and microperoxidase. Their purity and binding to serum proteins.
Authors: Bundgaard M , Møller M
Published: April 2011

Buffer solutions of commercially available batches of the electron microscopic enzyme tracers, horseradish peroxidase (HRP, mol wt = 40,000) and microperoxidase (MP, mol wt = 2,000), were investigated by gel filtration. The elution profiles of the HRP and MP solutions together with estimates of the peroxidatic activity of the collected fractions indicated that HRP was homogeneous with respect to molecular weight, while MP contained high molecular weight impurities with peroxidatic activity. Serum samples from four different species of tracer-injected animals (hagfish, lamprey, frog, mouse) and from control animals were investigated with the same techniques. No binding between HRP and the serum proteins or degradation of HRP was detected; whereas, MP circulated predominantly bound to serum proteins in all the investigated animals. The number average molecular weights of aqueous solutions of HRP and MP determined by vapor osmometry were 9,000 and 1,000, respectively. The results emphasize that protein binding should be evaluated when MP is used as an electron microscopic tracer. In addition, the finding that HRP and MP solutions have a much higher osmotic activity than the molecular weights of the tracers would suggest should be taken into consideration, especially when bolus injections into animals are performed.
Localization of DNA phosphorus in Tipula iridescent virus by oxygen plasma microincineration. II. Electron probe X-ray microanalysis of ash constituents.
Authors: Thomas R S , Corlett M I
Published: April 2011

Microincinerated, 1-micrometer sections of Tipula iridescent virus (TIV) crystals were analyzed quantitatively with a wave-length-dispersive electron microprobe to determine the chemical composition of high-resolution ash patterns previously obtained from ultrathin sections of the same specimen (Thomas RS: J Histochem Cytochem 29:379, 1981). Parallel analyses were performed on intact sections. In some cases, the same section and probe tracks were analyzed both before and after ashing. The principal elemental constituent of the ash was phosphorus, representing nearly all of the phosphorus found in the unashed sections. This confirmed the likely DNA origin of most of the ash. Other elements--sulfur, calcium, sodium, and perhaps carbon and nitrogen--found in relatively small concentrations in the ash, were partly due to either to incomplete ashing or to a particulate contaminant. Similar plasma ashing and analyses of sectioned model preparations of polymethacrylate containing dissolved triphenyl phosphate confirmed that phosphorus, by itself, could be retained as a stable ash. Analyses of polymethacrylate containing aluminum as well as phosphorus disclosed an unexpected artifact--aluminum inhibited the plasma ashing. These results suggested that the wavelength-dispersive probe, able to analyze for carbon and nitrogen as well as mineral elements, should be a generally useful tool in analyzing plasma microincineration phenomena, where macroscopic results do not apply. Relatively high beam intensity used throughout the probe analyses caused obvious damage to intact sections, particularly when they were mounted on thin-film supports. In contrast, the ash appeared quite stable. This suggested that plasma ashing of biological sections, converting them into mineralogical specimens, may be generally useful in probe analyses of mineral constituents, permitting greater sensitivity via higher beam currents, higher mineral concentrations, and lower backgrounds.
Staining patterns for the anti-horseradish peroxidase antibody reaction in proplasma cells developing in the medulla of rat popliteal lymph nodes during the secondary response.
Authors: Straus W
Published: April 2011

The development of plasma cells from lymphocytes was studied in the medulla of popliteal lymph nodes of rats during the secondary response to horseradish peroxidase (HRP). Changes in the microscopic appearance of proplasma cells were compared with changes in the intensity of the anti-HRP antibody reaction in these cells. Early proplasma cells, appearing 2 to 3 days after the injection of HRP into the footpads, were relatively small cells similar in size to lymphocytes. Their small nuclei were eccentrically located due to the one-sided enlargement of the pyroninophilic cytoplasm. The reaction for the anti HRP antibody in these cells was weak or negative. Other proplasma cells located in the same medullary cord regions showed a more intense antibody reaction. This change was correlated, in many cases, with an enlargement of the nucleus, giving the cells a blast-like appearance. Three to 6 days after the reinjection of the antigen, the medullary cords contained many mature plasma cells characterized by an intense antibody reaction. The mature plasma cells were always accompanied by proplasma cells, the latter varying in microscopic appearance (stage of development) asd staining intensities (antibody contents). The staining intensities and the microscopic appearance of proplasma cells, and the proportion of proplasma cells to plasma cells, varied in different medullary cord regions of the same lymph nodes. The staining patterns, together with the microscopic appearance of the cells, seemed to show whether antibody formation was inhibited or stimulated.
Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures.
Authors: Hsu S M , Raine L , Fanger H
Published: April 2011

The use of avidin-biotin interaction in immunoenzymatic techniques provides a simple and sensitive method to localize antigens in formalin-fixed tissues. Among the several staining procedures available, the ABC method, which involves an application of biotin-labeled secondary antibody followed by the addition of avidin-biotin-peroxidase complex, gives a superior result when compared to the unlabeled antibody method. The availability of biotin-binding sites in the complex is created by the incubation of a relative excess of avidin with biotin-labeled peroxidase. During formation of the complex, avidin acts as a bridge between biotin-labeled peroxidase molecules; and biotin-labeled peroxidase molecules, which contains several biotin moieties, serve as a link between the avidin molecules. Consequently, a "lattice" complex containing several peroxidase molecules is likely formed. Binding of this complex to the biotin moieties associated with secondary antibody results in a high staining intensity.
Ultrastructural localization of nuclei acids by the use of enzyme-gold complexes.
Authors: Bendayan M
Published: April 2011

A cytochemical technique for the ultrastructural localization of substrates using enzyme-gold complexes is reported. RNase A and DNase I have been labeled with gold particles. The RNase-gold and dNase-gold complexes obtained were applied on thin sections of glutaraldehyde-fixed and Epon-embedded tissues. Different cellular compartments were labeled by these enzyme-gold complexes. Using the RNase-gold complex the rough endoplasmic reticulum appeared decorated with gold particles. The gold marker was also present over the nucleus, especially over the nucleolus; mitochondria were weakly labeled. Using the DNase-gold complex, gold particles were concentrated over the euchromatin of the nucleus and the mitochondria. The heterochromatin and the nucleolus showed a less intense labeling. For both enzyme-gold complexes, the Golgi area, the secretory granules and the extracellular space appeared free of label. In those control conditions where the substrates were added to the enzyme-gold complexes a major reduction in the labeling was observed. A quantitative evaluation of the labeling was performed. This evaluation confirmed the qualitative observations and the marked reduction of labeling occurring under the control conditions. The combination of the specificity of the enzyme-substrate interactions with the size and electron density of the gold particles and the good ultrastructural preservation of the tissues resulted in a very specific labeling with high resolution. These results demonstrate the possibility of detecting substrates by means of enzyme-gold complexes at the electron microscope level.
Uptake of horseradish peroxidase by presynaptic terminals of bipolar cells and photoreceptors of the from retina.
Authors: Evans J A , Liscum L , Hood D C , Holtzman E
Published: April 2011

The uptake of horseradish peroxidase (HRP) into synaptic vesicles of presynaptic terminals in the inner and outer plexiform layers of isolated frog retinas was studies by electron microscopy. Uptake into the terminals of bipolar cells was found to be enhanced by exposure of the preparations to elevated concentrations of potassium ions, and by exposure to aspartic acid or glutamic acid. Glycine had much less effect on the terminals. These results suggest that HRP uptake may prove useful in monitoring some aspects of the responses of inner plexiform layer cells to conditions of physiological interest. Uptake into photoreceptor terminals was also enhanced by elevated potassium concentrations; the effects of the amino acids were complex.
Immunocytochemical detection of B cells in the guinea pig pancreas using guinea pig antiserum to porcine insulin.
Authors: Baskin D G , Gorray K C , Fujimoto W Y
Published: April 2011

The purpose of this study was to determine if antisera to an heterologous insulin could replace specific anti-guinea pig insulin serum for immunoperoxidase localization of guinea pig B cells. We found that antisera to porcine insulin was effective for this purpose even though it was produced by guinea pigs. The immunostaining results obtained with anti-porcine insulin sera and anti-guinea pig insulin serum were identical, by both light and electron microscopic immunoperoxidase staining. Control experiments supported the conclusion that the guinea pig antisera to porcine insulin contained antibodies that reacted with guinea pig insulin.
The effect of epidermal growth factor (EGF) on the differentiation of the rough endoplasmic reticulum in fetal mouse small intestine in organ culture.
Authors: Beaulieu J F , Calvert R
Published: April 2011

The differentiation of the rough endoplasmic reticulum (RER) of mouse duodenal absorptive cells located at the tip of the villi at 17 days of gestation was compared to that of absorptive cells in duodenal explants of 15-day-old mouse fetuses cultured for 72 hr 1) with Trowell T8 medium (without insulin) alone or supplemented 2) with epidermal growth factor (EGF; 100 ng/ml) or 3) with 25% bovine amniotic fluid (BAF). Glucose-6-phosphatase activity (G6Pase) was localized cytochemically to ensure a better identification of the RER. The intersections of a double lattice falling over and outside the RER were counted and the percentage of intersections over the RER was estimated. With this method, the extent of the RER is not statistically different when the absorptive cells in utero are compared to those of explants cultured with EGF. However, the extent of the RER in the absorptive cells cultured with Trowell T8 medium alone or supplemented with BAF is 50% lower than in the former two groups. It is concluded that EGF promotes the maturation of duodenal absorptive cells in organ culture.
Quantitative distribution of choline acetyltransferase and acetylcholinesterase activities in the rat cochlear nucleus.
Authors: Godfrey D A , Matschinsky F M
Published: April 2011

Within the cochlear nucleus of the rat, as well as some nearby regions, quantitative histochemical mapping procedures were used to construct maps of the distributions of choline acetyltransferase and acetylcholinesterase activities. The results were in some ways consistent with results previously reported for cat, e.g., very low activities of both enzymes were found in the auditory nerve root, and also in the vestibular nerve root, except where acetylcholinesterase-positive centrifugal fibers are located, very high activities were found in the facial nerve system. In many ways, however, the results for the rat cochlear nucleus contrasted with those for the cat. Notably, choline acetyltransferase activities in some regions of the rat cochlear nucleus were as much as 30- to 60-fold higher than for the comparable regions in the cat, and both enzymes had much more uniform distributions in the rat cochlear nucleus than in the cat. The more prominent cholinergic system in the rat cochlear nucleus might relate to a proportionately larger population of cholinergic interneurons, or, more probably, a more significant innervation by cholinergic, centrifugal pathways, or both, as well as, perhaps, generally higher choline acetyltransferase activities in cholinergic neurons of rat.
DNA analysis and sorting of viable mouse testis cells.
Authors: Grogan W M , Farnham W F , Sabau J M
Published: April 2011

The dye Hoechst 33342 and a 2-parameter cell sorter have been used to measure DNA content in viable testis cells and to sort pachytene spermatocytes and round spermatids from adult mouse testis to virtually 100% homogeneity. Early diploid spermatogenic cells were enriched to 90%, a 10-fold purification. The capability for viable sorting of most testis cell types to homogeneity in numbers suitable for many biochemical applications is demonstrated.
Immunohistochemical localization of procollagens. III. Type I procollagen antigenicity in osteoblasts and prebone (osteoid).
Authors: Wright G M , Leblond C P
Published: April 2011

Frozen sections of unfixed tibia and mandibles from day-old rats were immunostained by the peroxidase-antiperoxidase technique after exposure to antisera against procollagen I or other collagenous materials. Light microscopic study of bone growth areas showed that procollagen I antigenicity was present in osteoblasts and prebone (osteoid), but not bone tissue; neither procollagen III nor collagen IV antigenicity were detected. The localization of procollagen I antigenicity within osteoblasts was then attempted in the electron microscope. Chopper slices of formaldehyde-fixed tibia from day-old rats were incubated with affinity-purified anti-procollagen I antibodies linked to peroxidase and were treated with hydrogen peroxide in the presence of 3,3'-diaminobenzidine. The dot-like reaction indicative of procollagen I antigenicity was found to be moderate in rough endoplasmic reticulum (rER) cisternae, strong in spherical and cylindrical Golgi distensions, and intense in prosecretory and secretory granules. Reactivity was also observed within the matrix of multivesicular bodies Thus an increasing gradient of procollagen I antigenicity occurs from rER cisternae through Golgi distensions to secretory granules, that is, along the presumed pathway of procollagen synthesis. The antigenicity present within rER cisternae is attributed to the precursor pro alpha (I) chains, while that in the cylindrical Golgi distensions, secretory granules, and prebone is attributed to procollagen itself. The antigenicity of multivesicular bodies suggests some degradation of pro alpha chains or procollagen.
Immunoperoxidase staining of primate pituitaries with antibodies against the beta subunits of human pituitary glycoprotein hormones.
Authors: Halmi N S
Published: April 2011

The immunocytochemical specificity of antibodies against the beta subunits of human pituitary glycoprotein hormones was tested on human and monkey hypophyses. Anti-luteinizing hormone (LH) beta stained only gonadotrophic cells in both species, and anti-thyroid-stimulating hormone (TSH) beta only thyrotrophic cells. In monkeys, anti-follicle-stimulating hormone (FSH) beta demonstrated only gonadotrophs, but in man the antibody stained thyrotrophs equally well. Staining of the two cell types was diminished to a similar degree by dilution of the anti-FSH beta antibody. Human gonadotroph heterogeneity was revealed by absorption of anti-FSH beta with LH beta or TSH beta, which abolished immunostaining of a subset of gonadotrophs. Limitations in the immunocytochemical specificity of antibodies against beta subunits of pituitary glycoprotein hormones may be a consequence of incomplete purity of the generating antigens, structural overlap among the beta subunit of the three hormones, or a combination of these.
Probability paper analysis of flow cytofluorometric measurements of cellular deoxyribonucleic acid content.
Authors: Neely J E , Combs J W
Published: April 2011

A graphic analysis of cellular deoxyribonucleic acid (DNA) histograms obtained by flow cytofluorometry is described. This technique utilizes probability paper that graphs data in cumulative percentile form. From this graphic representation, individual normal distributions contributing to the histogram may be isolated and statistically describe. G0G1 and G2M distributions are independently described and S phase is fit to 1--5 overlapping normal distributions as dictated by the analysis results. This technique offers several advantages, including relatively simple mathematical calculations and few assumptions or restraints placed on the analysis. It has proved to be useful in analyzing proliferating and nonproliferating populations as well as distributions with unusual findings such as debris or aberrant DNA contents.
Discrete Distribution of binding sites for Dolichos biflorus agglutinin (DBA) and for peanut agglutinin (PNA) in mouse organ tissues.
Authors: Watanabe M , Muramatsu T , Shirane H , Ugai K
Published: April 2011

The distribution of binding sites for two lectins with different specificities was studied in adult mice by staining paraffin sections with lectins labeled either with fluorescein isothiocyanate or horseradish peroxidase. Binding sites for Dolichos biflorus agglutinin, a lectin specific to terminal alpha-N-acetylgalactosamine residue, were detected only in several restricted regions, such as collecting tubules and Bowman's capsules of the kidney, bile ducts, pancreatic ducts, sperms, oocytes, some secreting cells, and the secreted mucin itself. Binding sites for peanut agglutinin (PNA), a lectin specific to terminal beta-galactosyl residue, were distributed more widely. However, differentiation-dependent alterations in the expression of PNA binding sites have been observed in two types of cell lineage. During the course of spermatogenesis, the binding sites were expressed at the stage of spermatocytes. In the epithelium of esophagus, the binding sites were present in cells of the superficial layer.
Immunoelectron microscopic localization of tyrosinase in the mouse melanocyte.
Authors: Yamamoto H , Takeuchi T
Published: April 2011

In the melanocyte, tyrosinase is known as the dey enzyme for melanin formation. Purified tyrosinase protein was prepared that was capable of oxidizing tyrosine. The localization of tyrosinase antigen in the melanocyte was studied using antiserum against tyrosinase. DOPA (L-3,4-dihydroxyphenylalanine)-reaction product and tyrosinase antigen were found on the same organelles i.e., premelanosomes, melanosomes, GERL, and Golgi vesicles. This result seems to suggest that it is cytochemically appropriate to use DOPA as the substrate of tyrosinase. It appeared that tyrosinase antigen was present as granule-like structures inside GERL cisterna and associated with its membrane.
Decrease of glutamate decarboxylase (GAD)-immunoreactive nerve terminals in the substantia nigra after kainic acid lesion of the striatum.
Authors: Oertel W H , Schmechel D E , Brownstein M J , Tappaz M L , Ransom D H , Kopin I J
Published: April 2011

Antiserum sheep 3 against rat brain glutamate decarboxylase (GAD) was employed for the immunohistochemical localization of GAD-immunoreactive nerve terminals in the substantia nigra (SN). To test whether the antiserum specifically localized GAD-containing axon terminals, the effect of kainic acid-induced striatal lesions on the reactive nerve endings in the SN was investigated. Seven days after the injection of 1 microgram kainic acid into the striatum, a 65% decrease in GAD-enzyme activity occurred in the ipsilateral SN. On immunohistochemical examination there was correspondingly a marked reduction of GAD-positive terminals. The parallel decrease in biochemical and immunohistochemical GAD activity indicates that antiserum sheep 3 can be used as a specific immunohistochemical probed for GAD-containing elements.
Immunocytochemistry of secretory peptides: immunochemical dissections of peptide diversity.
Authors: Larsson L I
Published: April 2011

Immunocytochemistry of secretory peptides involves the localization of hormonal, neuronal, and paracrine messenger molecules at the cellular level. Immunological studies of these peptides have revealed them to be heterogenous at both the cellular, subcellular, ontogenetic, phylogenetic, and molecular level. It is probable that this heterogeneity reflects a diversity in function and that the same peptide backbone may be utilized for different tasks by different cells. One tentative way in which this could be brought about is by selective proteolytic, processing mechanisms. Although preliminary evidence indicates that such site-dependent processing occurs, much work remains to be done on the accurate localization of peptide fragments by specific, precise, sensitive, and selective tools. Recent progress in antibody production and evaluation as well as in immunocytochemical methodology has put such tools at our disposal. The present review discusses the use of these tools with particular emphasis on the interpretation of results, and their correlation to chemical and physiological information.
HISTOCHEMICAL DEMONSTRATION OF ACID INORGANIC POLYPHOSPHATASE
Authors: BERG GEORGE G.
Published: April 2011

A substrate solution for histochemical staining of acid inorganic polyphosphatase is prepared by controlled precipitation at pH 3.9, from ingredients totaling .014 M. lead, .05 M. acetate, .02 M. phosphate in the form of Graham's salt, and sufficient nitric acid to titrate. The final concentration of polyphosphate in solution is estimated at 5 x 10–4 M. in terms of total phosphate.

Inorganic polyphosphatase is stainable after a variety of fixation methods and paraffin embedding, but not after treatment with 70% and 80% ethyl alcohol or with excess substrate. The presence of a divalent metallic cofactor, which can be reversibly removed by water, is indicated.
A MODIFICATION OF GOMORI'S METHOD FOR DEMONSTRATION OF PHOSPHAMIDASE IN TISSUE SECTIONS
Authors: MEYER JULIA , WEINMANN J. P.
Published: April 2011

The shortcomings of Gomori's method for the histochemical demonstration of the enzyme phosphamidase lie in the need for a long incubation period and for the distinction between reaction products due to enzyme action and those due to spontaneous hydrolysis of the substrate. A modification of his method is suggested which permits reduction of the incubation period to about a fourth and eliminates the need to distinguish between reaction products of different origin in brief incubations.

The suggested procedure is free from erratic failures to obtain positive results. As tested for brain, the overall pictures of enzyme distribution are uniformly reproducible. Variability of results is reduced to the occasional occurrence of small areas in which no precipitate has formed. It is felt that with these changes the method is ready to be subjected to a more advanced stage of investigation, as for instance the preservation of the enzyme content during fixing, dry-freezing, etc.
FACTORS AFFECTING THE ACTIVITY OF ADENOSINE TRIPHOSPHATASE AND OTHER PHOSPHATASES AS MEASURED BY HISTOCHEMICAL TECHNIQUES
Authors: PADYKULA HELEN A. , HERMAN EDITH
Published: April 2011

Higher phosphatase activity toward ATP and other substrates occurred at alkaline pH in thin (5 µ), unfixed, frozen sections incubated in the medium described by Gomori ('41) than when treated with the medium employed by Maengwyn-Davies et al ('52). This difference in activities was caused principally by the salt content of the media. High sodium acetate concentration was strongly inhibitory to phosphatase activity toward adenosine triphosphate and α, β sodium glycerophosphate

The pH optimum of phosphatase activity of frozen sections toward adenosine triphosphate varied with the composition of the incubating medium. The localization of phosphatase activity toward adenosine triphosphate in cardiac muscle varied with substrate concentration, with nuclear staining becoming more prominent with increased adenosine triphosphate concentrations. The localization of phosphatase activity was not affected by the concentrations of α,β sodium glycerophosphate and adenosine-5-phosphate used, but the most rapid staining occurred with the highest concentrations.
PROBLEMS IN QUANTITATING RENAL ENZYMES
Authors: LONGLEY J. B. , BAHN R. C.
Published: April 2011

Histochemical assay methods for renal dehydrogenase present both biochemical and morphological problems. In each case practical solutions for these problems have been found. By a combination of simple procedures it is possible to derive an estimate of succinic dehydrogenase activity of the major reactive histological components of the kidney. The general approach should be useful in the future study of the response of renal dehydrogenase to experimental procedures.
CYTOPHOTOMETRIC DETERMINATION OF PROTEIN-BOUND SULFHYDRYL GROUPS
Authors: CAFRUNY E. J. , DI STEFANO H. S. , FARAH A.
Published: April 2011

The method of Barrnett and Seligman for histochemical visualization of protein-bound SH groups in tissue cells has been modified for relative quantitative use. Comparison with the Bennett method of sulfhydryl staining shows that the methods are comparable with respect to localization of sulfhydryl staining. The experimental results reported above indicate that the Barnett-Seligman method will satisfy major requirements for quantitative histochemistry.
UPTAKE OF RADIOACTIVE SULFATE BY ARTERIES OF NORMAL AND CHOLESTEROL-FED RABBITS
Authors: BUCK ROBERT C.
Published: April 2011

1. Normal and cholesterol-fed rabbits were injected intravenously with 0.6 to 1.0 mc. of S35 (as Na2SO4) and the distribution of the isotope studied by means of autoradiographs. The distribution of S35 was compared with that of stainable mucopolysaccharide.

2. In the normal aorta and pulmonary arteries a close parallelism is seen between the distribution of S35 and that of mucopolysaccharide.

3. The media of the atheromatous aorta, compared to that of the normal aorta, shows a reduced mucopolysaccharide staining and also a reduced isotope incorporation.

4. The intimal lesions show a high mucopolysaccharide content and also a high concentration of isotope, as compared with the media of normal or atheromatous aortas.

5. Fibroblasts and foam cells are situated in relation to the areas of greatest density in the autoradiographs. The deeper necrotic parts of the intima show little S35 incorporation although they contain stainable ground substance.

6. It is suggested that foam cells, as well as fibroblasts, produce the sulfated ground substance.
THE HISTOCHEMICAL DEMONSTRATION OF ACID PHOSPHATASE BY A POST-INCUBATION COUPLING TECHNIQUE
Authors: RUTENBURG ALEXANDER M. , SELIGMAN ARNOLD M.
Published: April 2011

The procedure for the histochemical demonstration of acid phosphatase by a post-incubation coupling technique using sodium 6-benzoyl-2-naphthyl phosphate is described. The distribution of activity in the tissues of the rat is presented. Enzymatic activity was generally confined to the cytoplasm of epithelial cells. Prostate, liver, spleen, ovary and kidney showed the greatest acid phosphatase activity.
FURTHER STUDIES ON THE HISTOCHEMISTRY OF GLUCOSE-6-PHOSPHATASE
Authors: CHIQUOINE A. DUNCAN
Published: April 2011

The tissues of the mouse were surveyed for glucose-6-phosphatase activity and the enzyme was found to be present only in the liver, kidney, small intestine, and rectum. The amount and distribution of the enzyme in the liver was not perceptibly altered in adrenalectomized animals or animals with alloxan diabetes. Some increase in activity was appreciated in animals starved for 72 and 96 hours. When sections were incubated in the presence of insulin or dextrose, no activation or inhibition was observed. Alloxan, on the other hand, did partially inhibit the enzyme. The action of fluoride, zinc, magnesium, and cyanide ions was also investigated. The possible role of the enzyme in the transport of glucose across cell membranes, involving phosphorylation and dephosphorylation, is discussed.
HISTOCHEMISTRY OF HUMAN ESTERASES
Authors: GOMORI GEORGE
Published: April 2011

Enzymatic distribution patterns of human esterases were studied by the use of a number of substrates and of activator and inhibitor substances. On the basis of their sensitivity to effector substances, human esterases can be classified in three large groups: hepatic, pancreatic and general type. In occasional cases, differential properties of esterases may be of some diagnostic value.
Double immunocytochemical labeling applying the protein A-gold technique.
Authors: Bendayan M
Published: April 2011

In the present study we report the modifications and the different steps of the protein A-gold (pAg) technique that allow the simultaneous demonstration of two antigenic sites on the same tissue section. The labeling is carried out in the following manner: face A of the tissue section is incubated with an antiserum followed by a pAg complex prepared with large gold particles; face B of the same tissue section is then incubated with a second antiserum followed by a pAg complex prepared with small gold particles. Each of the pAg complexes reveals a different antigenic site on opposite faces of the tissue section. The transparency of the section in the electron beam allows the visualization of the gold particles present on both faces. The double labeling pAg technique was applied for the simultaneous demonstration of two secretory proteins in the same Golgi, condensing vacuoles, and zymogen granules of the rat pancreatic acinar cells.
Histochemical and ultrastructural characterization of subendothelial glycoprotein microfibrils interacting with platelets.
Authors: Birembaut P , Legrand Y J , Bariety J , Bretton R , Fauvel F , Belair M F , Pignaud G , Caen J P
Published: April 2011

The interaction of human blood platelets with collagenase-treated rabbit subendothelium was studied by histochemical ultrastructural methods and by morphometric semi-quantitative analysis. Aortas were deendothelialized and incubated: 1) with a highly purified bacterial collagenase whose specificity was controlled; and 2) with the same collagenase followed by chymotrypsin. For histochemical studies, tannic acid, ruthenium red, and peroxidase-labeled Ricinus communis and concanavalin A were used. Electron microscopy showed that after digestion of fibrillar collagen by collagenase, adherent and aggregated platelets were observed on Ricinus communis-, concanavalin A-, and ruthenium red-positive glycoprotein microfibrils. After successive incubation with collagenase and chymotrypsin, the microfibrils disappeared. No platelets were observed on the remnant amorphous elastin. Morphometric analysis confirmed the interaction of platelets with collagenase-treated subendothelium. In addition, glycoproteins were extracted from collagenase-treated rabbit aortas using 5 M guanidine. Using an in vitro quantitative test, significant platelet adhesion to these glycoproteins was observed. Our results show an interaction between platelets and noncollagenic glycoprotein microfibrils.
Aluminum phosphate visualisation of acid phosphatase activity: a biochemical and x-ray microanalysis study.
Authors: Berry J P , Hourdry J , Sternberg M , Galle P
Published: April 2011

A new method is described that demonstrates acid phosphatase activity in the cells of the proximal tubules of the rat kidney. The method is based on the formation of an insoluble aluminum phosphate precipitate. Microanalysis was used to demonstrate the presence of intracellular aluminum and determine the quantity present under the probe. Parallel biochemical studies showed that the aluminum precipitate was indeed due to acid phosphatase activity.
Immunohistochemical localization of human pituitary glycopeptide (HPGP)-like immunoreactivity in the hypothalamus and pituitary of normal and homozygous diabetes insipidus (Brattleboro) rats.
Authors: Lu C L , Cantin M , Seidah N G , Chrétien M
Published: April 2011

Sections of the hypothalami and pituitary glands of normal (Sprague-Dawley) and homozygous diabetes insipidus (Brattleboro) rats were stained with antiserum to a human pituitary glycopeptide (HPGP) by using the immunohistochemical peroxidase-antiperoxidase method at the light microscopic level. Our results show in normal rats that immunoreactive HPGP was localized in the perikarya of the magnocellular neurons of the hypothalamus, in the posterior pituitary, and in the nerve fibers distributed in the median eminence (ME) and in the areas between the supraoptic nuclei (SON), paraventricular nuclei (PVN), and median eminence and also in the suprachiasmatic nuclei (SCN), a part of the parvocellular system. In the Brattleboro rats, however, no staining was found either in the hypothalami or pituitary glands. The present data strongly support our previous hypothesis that HPGP, a 39 residue glycopeptide isolated from human neurohypophysis, may be part of the precursor of arginine-vasopressin and its neurophysin II (Pro-NP-AVP).
Autoradiographic localization of steroid binding in human tissue labeled in vitro.
Authors: Shannon J M , Cunha G R , Taguchi O , Vanderslice K D , Gould S F
Published: April 2011

A procedure for the rapid preparation of autoradiograms from tissues incubated in vitro with 3H-estradiol is described. Slices of tissue were incubated in culture medium containing 17 nM 3H-estradiol, washed to remove unbound steroid, then processed for thaw-mount autoradiography. Exposure times were generally 3 to 4 weeks. Simultaneous in vitro competition with unlabeled progesterone, dihydrotestosterone, or hydrocortisone had no effect on the distribution or intensity of exposed silver grains, while competition with unlabeled estradiol or moxestrol abolished nuclear localization of silver grains. The exchange of labeled estradiol for bound endogenous estradiol during in vitro incubation of the tissues was demonstrated by a comparison of the pattern of incorporation of 3H-estradiol in tissues previously treated in vivo with unlabeled estradiol versus those not primed. A similar distribution and intensity of silver grains was observed in both the treated and untreated tissue groups. The rationale for the advantages of in vitro steroid autoradiography versus the in vivo technique is discussed.