bluuebunny/crossref_metadata_2025 huggingface dataset:
Light microscopic, immunohistochemical localization of the pia-glial basal lamina.
Authors: Kowalski T F , Vahlsing H L , Feringa E R
Published: April 2011

The current histologic methods for studying the pia-glial basal lamina (BL) are inappropriate for high contrast, permanent light microscopy preparation. We have developed a staining technique for epithelial BL which is highly specific, extremely sensitive, permanent, relatively inexpensive, and suitable for light or electron microscopy (EM). Central nervous system (CNS) basement membrane zone (BMZ) antigens were isolated by the technique of Meezan (1975) from female albino Wistar rats. Using this CNS BMZ preparation as an antigenic source, a hyperimmune rabbit serum was developed. This serum was exhaustively adsorbed with rat splenic pulp to remove undesirable antibodies to endothelial BL and collagen. The peroxidase-antiperoxidase indirect antibody technique was used to test the staining specificity of this splenic adsorbed serum on different tissues containing BLs of known origin and/or function. The results indicated that this BL staining technique was specific for epithelial BL of the rat and of some other species.
Alkaline phosphatase and an acid arylamidase as marker enzymes for normal and transformed WI-38 cells.
Authors: Dolbeare F , Vanderlaan M , Phares W
Published: April 2011

A survey of eleven enzyme activity levels in normal and SV40 transformed (VA-13) WI-38 cells revealed that the transformed cell enzymes differed by a quantitative and qualitative change of alkaline phosphatase and a quantitative loss of an arylamidase. Alkaline phosphatase activity was found to be elevated in the transformed cells at confluency but not in log phase cultures. This elevated activity was heat stable, L-homoarginine resistant and L-phenylalanine sensitive and is probably the term placental isoenzyme. In nontransformed WI-38 cells, the alkaline phosphatase was heat labile, L-homoarginine sensitive and L-phenylalanine resistant and so is probably the liver isoenzyme. While the arylamidase activity from both normal and transformed WI-38 cells had identical pH optima and Km values, the activity was approximately 20 times higher in confluent WI-38 cells than in confluent VA-13 cells. Cytochemical staining techniques for both activities are described that permit identification of fluorescent product within the cells, analysis of activity levels, and separation of cells with high and low activities. Mixtures of WI-38 cells and VA-13 cells separated by flow cytometry on the basis of arylamidase activity were subsequently evaluated for alkaline phosphatase isoenzyme and found to have been simultaneously separated into heat labile and heat stable samples.
A rapid method for embedding tissues for electron microscopy using 1,4-dioxane and Polybed 812.
Authors: Shearer T P , Hunsicker L G
Published: April 2011

A rapid method for embedding tissues for electron microscopy is described. This method, which can be completed within 5 hr, uses 1,4-dioxane as the final dehydrating agent and Polybed 812 as the embedding medium. Satisfactory preservation of cellular structures is consistently achieved with a variety of normal and diseased tissues. This method may be of particular value to diagnostic electron microscopy laboratories where time and simplicity are critical.
Methods for immunoelectron microscopy: Localization of antigens in rat kidney.
Authors: Sisson S P , Vernier R L
Published: April 2011

This study evaluated a variety of fixatives and methods of tissue preparation for application of the direct peroxidase-labeled antibody technique to rat kidney specimens. Tissue ultrastructure was most satisfactorily preserved and the antigens studied (rabbit IgG, human IgG, and rat Tamm-Horsfall protein (THP) were adequately preserved after brief fixation with 1% glutaraldehyde (15 min), a mixture of paraformaldehyde (1%) and glutaraldehyde (0.05%), or a paraformaldehyde, lysine, periodate fixative. Glycerol substitution was considered an important step which minimized ice crystal artifacts. Freezing and thawing were essential steps that facilitated adequate penetration of labeled antibody to specific antigenic sites. The distribution of injected rabbit IgG (anti-rat glumorular basement membrane (GBM) antibody) was predominately on the lamina rara interna and externa of the GBM. Injected aggregated human IgG was found primarily within the spaces between glomerular mesangial cells. Rabbit anti-rat THP was localized primarily on the infolding membranes of cells of the ascending thick limb of Henle. We suggest that the methods described may have wide application.
Projections from vasopressin, oxytocin, and neurophysin neurons to neural targets in the rat and human.
Authors: Sofroniew M V
Published: April 2011

Projections from vasopressin, oxytocin, and neurophysin neurons to neural targets were examined using the immunoperoxidase method. In the rat, neural target areas were found in portions of the limbic system, diencephalon, mesencephalon, brain stem, and spinal cord. In the human, only target areas in the brain stem and spinal cord were investigated. The projections to these targets derive from hypothalamic magnocellular vasopressin or oxytocin neurons, as well as from parvocellular vasopressin neurons of the suprachiasmatic nucleus. In neural target areas, axo somatic, as well as axodendritic, contacts are made. The findings suggest that these projections interact with other neurons, rather than release hormone into the bloodstream.
Total cellular RNA content: correlation between flow cytometry and ultraviolet spectroscopy.
Authors: Bauer K D , Dethlefsen L A
Published: April 2011

Total RNA content in Chinese hamster ovary and HeLa-S3 cells determined by ultraviolet spectroscopy is compared with the red fluorescence distribution of acridine orange-stained cells observed by flow cytometry. A correlation coefficient of 0.93 is obtained when these methods of estimating RNA content are compared after various RNAse treatments. These data suggest that acridine orange staining effectively quantitates total cellular RNA content when analyzed by flow cytometry, although DNA is also shown to contribute a low but significant background of red fluorescence.
Carbonic anhydrase, ultrastructural localization in the mouse gastric mucosa and improvements in the technique.
Authors: Sugai N , Ito S
Published: April 2011

The ultrastructural localization of carbonic anhydrase activity in mouse gastric mucosal cells as revealed by the cobalt bicarbonate histochemical method of Hansson has been made. In addition the effects of fixatives used for ultrastructural studies have been evaluated for reduction of carbonic anhydrase activity; exogenous erythrocyte carbonic anhydrase has been localized in tissues; acetazolamide and potassium cyanate inhibition of activity demonstrated; and an improved method for the osmication of reacted tissues for electron microscopy has been developed. The results indicate that the glutaraldehyde, formaldehyde, picric acid fixative, which retains about 5% of the original carbonic anhydrase activity, is distinctly better for histochemical studies than formaldehyde fixation, which retains about 32% activity. Acetazolamide at 10(-5) M consistently inhibits histochemical reaction, as does 20 mM KCNO, in the incubation medium. Exogenous carbonic anhydrase is readily visualized by the histochemical technique. Electron microscopy of gastric mucosa reacted for carbonic anhydrase activity indicates the focal deposition of the cobalt sulfide reaction product in the cores of microvilli lining the intracellular canaliculi, in the basal and lateral cell folds of parietal cells, and in the microvilli as well as the cytoplasm between mucous granules in the surface mucous cells. In addition, some reaction product was found in the mitochondrial cristae and in some nuclei and intercellular spaces.
Incorporation of 3H-leucine, 3H-lysine, and 3H-tryptophan during the cell cycle of Chinese hamster ovary cells: a flow cytometric analysis.
Authors: Blair O C , Roti Roti J L
Published: April 2011

Exponentially growing Chinese hamster ovary cells were pulse labeled with 3H-leucine, 3H-lysine, or 3H-tryptophan, fixed, and stained by either the acriflavine-Feulgen procedure or with fluorescein isothiocyanate (FITC). Protein content as determined by FITC fluorescence was representative of protein content determined biochemically by the method of Lowry. Utilizing a fluorescence-activated cell sorter, 3H-labeled cells were sorted according to their DNA or protein content and the incorporation of 3H-leucine, 3H-lysine, or 3H-tryptophan determined by liquid scintillation counting. The rates of 3H-leucine, 3H-lysine, and 3H-tryptophan incorporation increased with respect to increasing DNA content (G1, mid-S, G2+M). The rate of 3H-lysine incorporation increased continuously with increasing protein content, whereas the rates of 3H-leucine and 3H-tryptophan incorporation were constant initially with an increase in incorporation near mid-cycle followed by a slight decrease. Matrix algebra modeling of the increase in protein content suggests that 3H-lysine incorporation is consistent with a sigmoidal increase in protein content, however, 3H-leucine and 3H-tryptophan incorporation do not follow either the exponential, linear, or sigmoidal models. Matrix algebra simulation of the FITC protein distribution indicates that while the rate of protein accumulation is not linear, the exponential and sigmoidal models fit the experimental data equally well.
Ultrastructural localization of exogenous carbonic anhydrase activity in ileal absorptive cells of suckling rats.
Authors: Sugai N , Ito S
Published: April 2011

To test the resolution and reliability of Hansson's histochemical reaction for carbonic anhydrase (CAH) activity at the electron microscopic level, purified exogenous bovine, rabbit, and human B and C CAH was reacted for histochemical activity after uptake by suckling rat ileal absorptive cells and examined microscopically. The cobalt sulfide reaction product was found confined to the apical vacuoles, tubules, and vesicles as small as 30 nm in diameter and was confined within the limiting membrane of the endocytic system. The histochemical technique did not distinguish between the different types or sources of the CAH nor were differences in their enzymatic activity apparent. It was concluded that when the cobalt precipitation technique of Hansson is used to demonstrate exogenous CAH activity it results in the precipitation of the reaction product at or very near the site of the CAH molecule. In addition to the resolution of the technique to demonstrate enzyme activity, this study suggests that the ultrastructural localization of intrinsic CAH activity can be accepted with greater confidence.
A rabbit bone marrow model system for evaluation of cytotoxicity: characterization of normal bone marrow cell cycle parameters by flow cytometry.
Authors: Muirhead K A , Irons R D , Bruns R , Horan P K
Published: April 2011

Characterization of a rabbit model system for the study of cell cycle effects of myelotoxic agents in normal bone marrow is described. Cell cycle phase distributions are obtained by computer analysis of flow cytometric single cell DNA histograms. Comparison of marrow aspirates with marrow samples from sacrificed animals indicates that dilution of aspirates with peripheral blood is not significant. Aspiration of marrow from one bone does not affect the cell cycle distribution of unsampled bones. Hence, sequential aspirates of different bones in a single animal may be used as representative samples for further study of effects of myelotoxins on marrow proliferation and differentiation.
Fluorophosphate-sensitive esterases in mammalian liver: the radioautographic localization and measurement of fluorophosphate-reactive sites in adult rat liver.
Authors: Budd G C , Barnard E A , Porter C , Mattimoe S A
Published: April 2011

Fluorophosphate-reactive (FPR) sites in the adult male rat liver, tentatively identified as esterase active centers, were localized and measured using the combined techniques of quantitative electron microscope radioautography and morphometric analysis with the light and electron microscope. The FPR sites were measured in liver which had been prefixed by perfusion with 1.5% glutaraldehyde and reacted with 10(-4) M tritium-labeled diisopropyl fluorophosphate (3H-DFP). Under the experimental conditions 64-67% of the esterase activity in fresh liver was retained for reaction with the 3H-DFP, which is known to bind irreversibly to the active sites of certain esterases. In light and electron microscope radioautographs the developed silver grains were concentrated over the cytoplasm of hepatocytes. A low concentration occurred over erythrocytes. All other areas in the liver had a concentration of grains resembling the background concentration. Quantitative measurements of grain density in the electron microscope radioautographs revealed the highest density, after correcting for radiation spread, in cytoplasmic granules (mainly cytolysomes). The grain densities over the rough and smooth endoplasmic reticulum and associated structures were also equal to or above the average hepatocyte grain density. Due to the large fractional volume of endoplasmic reticulum per hepatocyte (58% of cell volume) and the fraction of the liver occupied by hepatocytes (79% of liver volume) the majority of FPR sites in the liver occurred in the rough and smooth endoplasmic reticulum and associated structures. The average numbers of FPR sites were calculated per micrometer3 of hepatocyte (5.0 x 10(5) sites/micrometer3) and per unit volume of each significantly labeled organelle. In addition, the number of FPR sites per hepatocyte (2.5 X 10(9) sites/cell), per cm3 liver (4.1 X 10(17) sites/cm3) and in the total liver of an average 100 g male rate (2.2 X 10(18) sites/total liver) were also calculated.
In vitro hippocampal uptake of tritiated serotonin (3H-5HT): A morphological, biochemical, and pharmacological approach to specificity.
Authors: Azmitia E C , Marovitz W F
Published: April 2011

The in vitro uptake of tritiated serotonin (3H-5HT) into hippocampal slices was measured in Ringer's solution (37 degrees C) containing pargyline, ascorbic acid, and dextrose. The specific uptake of 3H-5HT rose asymptotically as the 3H-5HT molarity was increased from 5 x 10(-10) to 1.5 x 10(-6) M. Linear regression analysis gave a Km value for the specific uptake of 1.4 x 10(-7) M. The nonspecific binding (NSB) was the amount of 3H-5HT retained by the slices following incubation in a medium with a very large excess of unlabeled 5-HT added to dilute the specific uptake of 3H-5HT. This NSB increased with increasing molarity of 3H-5HT, and was linearly related to 3H-5HT concentrations between 5 x 10(-9) and 1.5 x 10(-6) M. The ratio of specific uptake to NSB was highest at 5 x 10(-8) M (2.75) and lowest at 1.5 x 10(-6) M of 3H-5HT (0.54). Competition studies with noradrenaline, desipramine (a noradrenergic uptake blocker), fluoxetine (a 5-HT uptake blocker), and tryptophan confirmed the specificity of the 3H-5HT uptake mechanism. Radioautographic studies of in vitro incubated hippocampal slices showed silver grain aggregates at 3H-5HT specific uptake sites. Addition of an excess of unlabeled 5-HT to the slices, or the use of hippocampi from 5,7-dihydroxytryptamine intracerebral microinjected rats (5 microgram/400 nl into the fornix-fimbria and the cingulum bundle, 6 day survival) caused a dramatic decrease in these aggregates. The distribution of hippocampal 5-HT axons and terminals, inferred from the pattern of silver grain aggregates, is more widespread than previously described. 5-HT varicosities were clearly seen in all layers of Ammon's horn, dentate gyrus, and the subicular cortex. Innervation routes were seen to the stratum radiatum and stratum lacunosum from stratum oriens in Ammon's horn, and to the polymorphic layer of the dentate gyrus from the subicular cortex and from the fimbria. Semiquantitation of the occurrence of silver grain aggregates was done in the various hippocampal regions. The highest density in Ammon's horn was 119.5 boutons/10,000 micron2, in the dentate gyrus it was 67.4 boutons/10,000 micron2, and in the subicular cortex it was 79.2 boutons/10,000 micron2. These results are consistent with previous quantitative results.
Morphologic probes of polypeptide hormone receptor interactions.
Authors: Gorden P , Carpentier J L , Freychet P , Orci L
Published: April 2011

Polypeptide hormones, growth factors, and a variety of other naturally occurring ligands bind specifically to receptors on the cell surface. At physiologic temperatures these ligands are internalized by cells and associate intracellularly with lysosomes. Receptor-mediated internalization provides a simple mechanism that may act to couple receptor-mediated hormone degradation with receptor-medicated receptor regulation.
Immunohistologic analysis of the organization of normal lymphoid tissue and non-Hodgkin's lymphomas.
Authors: Stein H , Bonk A , Tolksdorf G , Lennert K , Rodt H , Gerdes J
Published: April 2011

Hoping to improve the systems for identifying and classifying normal and malignant lymphoid subpopulations, frozen and paraffin sections of nonmalignant lymphoid tissue and of malignant lymphomas were immunostained for surface (S) and cytoplasmic antigens using the peroxidase-antiperoxidase method. Primary follicle cells and follicle mantle cells known to be part of the recirculating B-cell pool were found to be constantly Ia and C3 receptor (C3R) positive, mostly SIgM and SIgD positive and cytoplasmic immunoglobulin (CIg) negative. The light zone of germinal centers (GC), which is rich in centrocytes, contained a large number of T cells and showed the well-known intercellular Ig network pattern; the dark zone, containing densely packed centroblasts, was usually free of T cells, but was bordered ay a mantle-like accumulation of T cells. Usually only some of the GC cells were definitely positive for SIg and CIg of different classes. All cells reacted positively for Ia and C3R. In areas described by other authors as containing marginal zone cells, cells densely bearing SIgM and deficient in SIgD were detected. The immunoblasts of the hyperplastic plasma cell reaction usually contained CIg. Cells from chronic lymphoid leukemia sections that immunostained for SIgM and SIgD were interpreted as representing a neoplasm of recirculating B cells expressing SIgM and SIgD. The immunohistologic architecture of follicular centroblastic/centrocytic lymphoma showed a more or less close similarity to the organization of secondary follicles. Lymphomas whose cells resembled reactive centrocytes were strongly SIgM positive and SIgD negative or only weakly SIgD positive. CIg was demonstrable in nearly 90% of the lymphomas whose cells resembled centroblasts and in 70% of the lymphomas whose cells resembled immunoblasts of the plasma cell reaction. Finally, immunohistologic staining results from a T-zone lymphoma are presented, which confirm that this lymphoma was composed of a neoplastic T zone and a non-malignant B zone.
Are histochemical methods for estrogen receptor valid?
Authors: Chamness G C , Mercer W D , McGuire W L
Published: April 2011

Because of the great usefulness of estrogen receptor determinations in selecting therapy for breast cancer patients a number of histochemical and immunohistochemical methods for visualizing bound estrogen in cells and tissue sections have been proposed. We discuss all of these histochemical methods in the light of the known properties of the estrogen receptor and other estrogen binders, and we consider some criteria that must be met if such methods are to be considered valid for receptor. In spite of the great potential value of histochemical methods, we are forced to conclude that, in their present form, none of them are likely as yet to be detecting estrogen receptor.
Histochemical analyses of steroid hormone receptors in breast and prostatic carcinoma.
Authors: Pertschuk L P , Tobin E H , Tanapat P , Gaetjens E , Carter A C , Bloom N D , Macchia R J , Eisenberg K B
Published: April 2011

Histochemical analyses estrogen (ER) and progesterone (PgR) receptors in breast cancer were statistically correlated with results of dextran-coated charcoal (DDC) and sucrose gradient assays. Correlated for ER was 91% of 363 cases, and for PgR 88% of 255 specimens. Breast cancer ER/PgR positivity by histochemistry correlated with a favorable clinical response to endocrine therapies in 72% of 25 cases, while ER/PgR negativity correlated with a lack of response in 96% of 22 cases with Stage IV disease. Nuclear ER/PgR correlated with a poor response to therapy in 8 of 12 patients. An in vitro technique to detect nuclear translocation of ER revealed two groups of ER positive cases, with 11 of 17 exhibiting translocation and 6 not displaying translocation. In prostatic carcinoma, 72% of 65 men were positive for ER and/or androgen receptor. Comparison of specimens obtained without and with electrocautery revealed a preponderance of nuclear binding in the latter, suggesting heat-induced nuclear translocation of receptor. coumestrol, a naturally fluorescent, entirely unaltered estrogen was also used for histochemical detection of ER. Results correlated with ER by DCC in 87% of 61 breast cancers. Coumestrol was additionally used to visually observe receptor and nuclear translocation of ER in intact whole cells in culture.
Influence of two different fixatives on the identification of plasma cells in human rectal mucosa.
Authors: Heyworth M F
Published: April 2011

Plasma cells in sections of bisected human rectal biopsy specimens, fixed in two alternative fixatives, were enumerated after staining by an indirect immunoperoxidase procedure intended to demonstrate immunoglobulin-containing cells. The counts of immunoperoxidase-positive plasma cells were significantly higher after fixation in formol sublimate than after fixation in formol saline. Formol sublimate appears to be a more reliable fixative than formol saline for specimens of rectal mucosa in which quantitation of plasma cells, stained for intracellular immunoglobulin by an immunoperoxidase technique, is intended.
Immunocytochemical localization of the vitamin D-induced calcium-binding protein: relocation of antigen during frozen section processing.
Authors: Taylor A N
Published: April 2011

The vitamin D-induced calcium-binding protein (CaBP) was localized in chick duodenum by the indirect fluorescent antibody technique after tissue was prepared by three different rapid-freezing methods: freeze-thaw, freeze-drying, and freeze-substitution. Sections prepared by freeze-thawing demonstrated CaBP-specific fluorescence over goblet cells and at the absorptive surface of villi, but not in absorptive cells. Sections from the same or adjacent segments prepared by freeze-drying or freeze-substitution produced virtually the opposite pattern of CaBP distribution. CaBP-specific fluorescence was associated with absorptive cell cytoplasm but not with goblet cells. From the results of experiments in which 6 micrometer freeze-dried sections were rehydrated, it was concluded that in the presence of an aqueous environment CaBP migrated from absorptive cells to discrete localization sites bound to goblet cell mucus, possibly to the calcium present in the mucus. By analogy, it was concluded that CaBP in association with goblet cells and the absorptive surface in sections prepared by the freeze-thaw method represented an artifactual localization. The true in situ localization of CaBP in chick duodenum was that which was present in absorptive cell cytoplasm.
"Fast" isomyosins and fiber types in mammalian skeletal muscle.
Authors: Pierobon-Bormioli S , Sartore S , Libera L D , Vitadello M , Schiaffino S
Published: April 2011

Immunohistochemical procedures have been used to correlate antigenic differences among fast isomyosins with their specific localization in particular types of rat muscle fibers. Antisera were produced in rabbits against myosins isolated from guinea pig fast-white (tensor fasciae latae) and fast-red (masseter) muscles. After sequential cross-adsorptions on insolubilized heterologous myosins, antibodies mainly directed against heavy chain determinants were obtained. In indirect immunofluorescence assays, these antibodies selectively stained fast-white (type IIB) and fast-red (type IIA) fibers, respectively. Slow-twitch (type I) fibers, which were reactive with antibodies against soleus muscle myosin, were unreactive with anti-fast-myocin antibodies. In addition to these major types of fibers, with unique immunoreactivity, two groups of fibers with double reactivity were identified: 1) fibers reactive with anti-slow-twitch and anti-fast-red antibodies (corresponding to IIC fibers with enzyme histochemistry) and 2) fibers reactive with anti-fast-red and anti-fast-white antibodies. We propose to identify these fibers with the symbols I in equilibrium with IIA and IIA in equilibrium with IIB, respectively, as they may be considered as intermediate and/or transitional stages between the major fiber types. No fibers were reactive with both anti-slow-twitch and anti-fast-white antibodies. Therefore, muscle fibers may change in a sequential manner, from I leads to IIA leads to IIB or in the reverse direction, suggesting an obligatory sequence of gene activation.
Ultrastructural localization of coenzyme A phosphatase (CoA-Pase) activity to the GERL system in secretory ameloblasts of the rat incisor.
Authors: Smith C E
Published: April 2011

Enzymatic hydrolysis of the monoester phosphate group from coenzyme A (CoA) was studied in rat incisor ameloblasts by incubating specimens from glutaraldehyde-fixed teeth in a cytochemical medium prepared with acetyl-CoA as substrate and lead ions as capture agent for phosphate. Ameloblasts incubated for 1 hr at 37 degrees C and at pH 5.0 in this medium showed reaction product localized almost exclusively along the trans (mature) aspect of the Golgi apparatus within a network of small granules and interconnecting tubular channels that comprise the GERL system in this cell. Reaction product was otherwise seen in trace amounts only within some Golgi saccules, a few lysosomal dense bodies and, in rare instances, within an occasional focal area of the endoplasmic reticulum. No selective staining of the GERL system was seen in control ameloblasts incubated at either pH 7.2 or pH 9.0 with acetyl-CoA as substrate, or incubated at pH 5.0 with dephospho-CoA as substrate. Control experiments at pH 5.0 also revealed that reaction product selectively stained the GERL system in ameloblasts when other molecules resembling CoA were used as substrate (e.g., crotonyl-CoA, 3'-NADP+), but not when adenosine 3'-monophosphate (3'-AMP) was used as substrate. That is, ameloblasts incubated at pH 5.0 with 3'-AMP showed heavy deposits of reaction product at many sites throughout the cell, including most lysosomal dense bodies, the Golgi saccules, the GERL system, most secretory granules, the nucleus, and extensively throughout the endoplasmic reticulum. These findings suggest that the GERL system of ameloblasts contains a CoA-specific phosphatase activity that may function to convert CoA to dephospho-CoA at acid pH. Biochemical studies included with this investigation further indicate that CoA-Pase activity saturates at exceptionally low concentrations of substrate (KM = 30 microM CoA) compared to other acid-dependent phosphatases.
Analysis of the changes in the morphology and staining density characteristics of cartilagenous cell nuclei from Triturus cristatus during aging and regeneration.
Authors: Jeanny J C , Gontcharoff M
Published: April 2011

The modifications in the morphology and staining intensity of cell nuclei reflect their physiologic and functional state. In order to appreciate the variations in the metabolic activity of hind limb chondrocytes from Triturus cristatus during aging and regeneration, we have studied the nuclear size and shape, the DNA concentration, and the distribution and degree of condensation of the chromatin. This study was carried out using scanning cytophotometry on semithin 1 micrometer sections, Feulgen-stained for DNA, which is the major nuclear component. During aging, the nuclei decrease in size, becoming irregular in shape. The concentration of DNA increases and the chromatin distribution becomes more heterogeneous with an increase in condensed chromatin and a subsequent decrease in the diffuse chromatin fraction. During regeneration, however, these modifications are reversed whatever the age of the animal, but even more noticeably when the animal is old. The changes that occur in the chromatin fractions defined according to their state of condensation are discussed from a functional point of view.
Tissue fixation and staining by osmium tetraoxide: a possible role for alkaloids.
Authors: Wright M J , Schröder M , Nielson A J
Published: April 2011

In vitro studies on the reactions of osmium tetraoxide, OsO4, with isoquinoline, pyridine, quinuclidine, and a series of structurally related alkaloids suggest that these species, under the conditions of normal tissue staining and fixation, are potentially osmiophilic. The structure of the products and their reactions with unsaturated substrates are described. The relevance of these results to the process of tissue staining and fixation of plant tissues containing alkaloids by OsO4 is discussed.
Relationship between peroxisomes and endoplasmic reticulum investigated by combined catalase and glucose-6-phosphatase cytochemistry.
Authors: Shio H , Lazarow P B
Published: April 2011

The theoretical advantages of electron microscopic cytochemistry were utilized to look for evidence of possible connections between peroxisomes and the endoplasmic reticulum in rat liver. Established cytochemical procedures for catalase (peroxisomes) and glucose-6-phosphatase (endoplasmic reticulum) were carried out, and evidence was sought of diffusion of reaction products between the organelles. No such diffusion was observed: lead phosphate was found in the endoplasmic reticulum and in the nuclear envelope but not in peroxisomes; oxidized diaminobenzidine (DAB) was seen only in peroxisomes. In addition, both types of cytochemistry were carried out on the same tissue. The two kinds of reaction product could be distinguished by virtue of their different electron opacities. No mixing of the two reaction products was observed. These results do not support the hypothesis that peroxisomes and endoplasmic reticulum may be connected; rather, they support the idea that the two organelles exist as separate cellular compartments.
Ultrastructural immunocytochemical localization of myosin in cultured fibroblastic cells.
Authors: Willingham M C , Yamada S S , Bechtel P J , Rutherford A V , Pastan I H
Published: April 2011

Nonmuscle myosin in the cytoplasm of cultured fibroblastic cells has been localized using light and electron microscopic immunocytochemistry. Antibodies to purified fibroblast myosin were produced in goat and rabbit and purified by affinity chromatography. Light microscopic immunofluorescence localization showed patterns similar to those previously published. Electron microscopic localization using the ethyldimethyl aminopropyl carbodiimide-glutaraldehyde-saponin (EGS) fixation-permeabilization procedure and the ferritin bridge localization method produced quantifiable localization in intracellular sites with well-preserved ultrastructural morphology. Myosin was found to be a major component of the cytosol. It was distributed diffusely with no preferential localization on membranous organelles. Myosin was found to be slightly concentrated on the surface of microfilament-containing structures, including the subplasmalemmal microfilament mat and stress fibers, occasionally with an interrupted periodicity. However, no myosin was found in surface ruffles or microvilli. Morphometric quantitation showed that the majority of the cell's myosin was in the cytosol. This location is compatible with myosin being a component of the microtrabecular lattice of the cytoplasmic ground substance. The concentration of myosin in association with microfilaments was only twice that of the cytosol. This interpretation must be somewhat tempered by the possibility that some myosin bound to tightly packed actin may be inaccessible. The significance of this distribution of myosin in cell function is discussed.
A simple fluorescent method for simultaneous determination of aortic permeability to horseradish peroxidase and bovine serum albumin.
Authors: Hollis T M , Katora M E , Montini J
Published: April 2011

Differences in regional aortic net uptake of bovine serum albumin (BSA) and horseradish peroxidase (HP) have been examined by means of conjugation of these molecules to the fluorescent protein tracers fluorescein isothiocyanate (FITC) and lissamine rhodamine B (RB200). Using male Wistar rats, uptake of FITC-BSA under steady state conditions in the ascending aorta and aortic arch (14 +/- 2 micrograms/mg aorta) is significantly higher (p less than 0.05) than that of either the upper, middle, or lower third of the thoracic aorta (10 +/- 1 mu, 9 +/- 1 mu, and 8 +/- 1 micrograms/mg, respectively). No regional variation in net uptake of RB200-HP was observed in these same aortic regions, with respective mean values (+/- SE) being 69 +/- 2, 69 +/- 2, 68 +/- 4, and 68 +/- 4 micrograms/mg. Examination of fluorescence photomicrographs indicate that FITC-BSA is localized along the collagen-elastin bands, while the RB200-HP is found between these bands. Differences between FITC-BSA and RB200-HP uptake and deposition reflect such things as differences in uptake rates as influenced by initial concentrations, permeability coefficients, as well as possible differences in molecular charge and affinity. The results indicate that the fluorometric procedures described in this investigation are simple, sensitive, and quantitative, and suitable for simultaneous measurement of aortic uptake of molecules having different molecular sizes as well as for the intraaortic localization of these substances.
Kallikrein in rat pancreatic tissue after beta cell destruction or acinar cell atrophy.
Authors: Orstavik T B , Brekke I B , Alumets J , Carretero O A
Published: April 2011

The purpose of this study was to determine whether glandular kallikrein in rat pancreas is located in the beta cells of the endocrine pancreas or in the acinar cells of the exocrine pancreas. Kallikrein was measured by radial immunodiffusion and a direct radioimmunoassay in homogenates of pancreas obtained from 1) control rats, 2) rats with pancreatic beta cells selectively destroyed by streptozotocin, and 3) rats with acinar cell atrophy induced by pancreatic duct occlusion. Beta cell destruction was confirmed by the presence of hyperglycemia and by an almost total depletion of insulin-producing cells as demonstrated immunohistochemically. Acinar cell atrophy was confirmed histologically and by an almost total depletion of trypsin-like enzymes in pancreatic homogenates. The concentration of kallikrein in pancreatic homogenates was unchanged after beta cell destruction, whereas it was greatly decreased following acinar cell atrophy. Kallikrein was, by immunohistochemistry, demonstrated in the acinar cell only. The immunohistochemical localization of kallikrein agrees with the above results. These studies strongly indicate that kallikrein is predominantly located in the acinar cells of the exocrine pancreas.
A method for the detection of abnormal leukocytes using cytochemical criteria.
Authors: Winkel P , Olesen T
Published: April 2011

An alarm system for the detection of abnormal leukocytes using off-line data processing of the image of the peroxidase channel oscilloscope picture of the Hemalog-D is presented. The basic idea is that areas on the oscilloscope picture where more than a negligible number of nonpathological leukocytes may be depicted are delimited from the remaining area, which is divided into three alarm zones. The corresponding alarm quantities are the large unstained cells (LUC) or the unstained alarm (UA), the intermediate alarm (IA), and the stained alarm (SA). Reference intervals for these alarms were established using blood specimens from 15 healthy subjects. The system was tested using blood specimens from four patients with acute lymphoblastic leukemia (ALL) and 11 patients with acute myeloblastic leukemia (AML). The UA was the best alarm overall, but for seven of the AML specimens the IA or the SA alarm was superior. The high peroxidase (HPX) and the remainder alarms were inferior to the other alarms. Using the reference mean plus two standard deviations as a cutoff value, the smallest blast cell number fraction detectable by the best alarm was calculated to be smaller than 2% for each of the AML specimens, while it ranged from 7 to 2.9% for the ALL specimens.
Quantitative data on peroxidatic markers for electron microscopy. With a note on actin identification in Paramecium cells.
Authors: Tiggemann R , Plattner H , Rasched I , Baeuerle P , Wachter E
Published: April 2011

Several important points of heme-peptide cytochemistry were quantitatively analyzed, with particular regard to their use in electron microscopic immunocytochemistry. A simple procedure is presented for the preparation of heme-octapeptide (H-8-P) microperoxidase. H-8-P, hemenonapeptide (H-9-P), and various horseradish peroxidase (HRP) isoenzymes were used for coupling with immunoglobulin (Ig)G or the papain-cleavage fragments from IgG (Fab) molecules. Ultracentrifugation and spectrophotometric analyses revealed the following characteristics of the conjugates: a) They are of a uniform size class; b) their diameters were calculated, and ranged from 5.6 (Fab-H-8-P; H-9-P) to 10.5 (IgG-HRP); c) the persistence of antigen binding capacity was ascertained; d) the deactivation of the marker peroxidase activity due to coupling was as low as 20-30%; e) optimal conditions for use of the electron microscopic (EM) with 3,3'-diaminobenzidine media were elaborated (with a pH optima somewhat different from some standard methods in current use); and f) on the basis of the quantitative data presented, an optimal compromise (either in favor of higher peroxidase activity with HRP conjugates or of smaller size with microperoxidase-Fab conjugates) can be achieved. Finally, the identification of isolated purified actin and of actin in cortical microfilament bundles and ciliary basal bodies of Paramecium cells served as a test object for the usefulness of conjugation products and optimized assay conditions for EM immunocytochemistry.
An improved histochemical method for distinguishing colonic acetylsialomucin from other epithelial mucins.
Authors: Cooper J H , Durning R G
Published: April 2011

A technical modification of the original histochemical methods for distinguishing human colonic acetylsialomucin from other epithelial mucins is described. The modification uses a naphthoic acid hydrazide-diazonium salt sequence in place of the original borohydride or blue Schiff treatments in the first stage of the staining procedure and a more dilute solution of periodic acid in the second stage. By these means, strongly contrasting staining of colonic acetylsialomucins in magenta red and all other mucins in dark blue may be consistently produced.
Autoradiographic Determination of Neurotransmitter Receptor Distributions in the Cerebral and Cerebellar Cortices,
Authors: Wamsley J.K. , Palacios J.M. , Young W.S. , Kuhar M.J.
Published: April 2011

Light microscopic autoradiographical localization of drug and neurotransmitter receptors provides investigators with a tool to determine the sites of drug action with a high degree of anatomical resolution. In the cortex, where there is a distinct laminar organization, the differential distribution of receptors can be determined. We have found that differences in the density of specific receptors exist between layers of the cerebral cortex. Our observations include the localization of muscarinic cholinergic, benzodiazepine, opiate, neurotensin, histamine-H1, alpha-adrenergic, beta-adrenergic, and high-affinity GABA receptors in the cerebral cortex. Of this list of neurotransmitters only significant concentrations of benzodiazepine, GABA, and beta-adrenergic receptors were found in the cerebellar cortex. Receptor localizations such as these can be coupled with information from immunohistochemical studies to provide the basis for future experimentation in order to define neuro-transmitter-specific pathways in the cerebral and cerebellar cortices.
Combined Autoradiography and Immunohistochemistry for Simultaneous Localization of Radioactively Labeled Steroid Hormones and Antibodies in the Brain,
Authors: Sar Madhabananda , Stumpf Walter E.
Published: April 2011

A combined technique of autoradiography and immunohistochemistry is described for localization of radioactively labeled ligands and antibodies to neuropeptides, protein hormones and neurotransmitter synthesizing enzymes in the brain. This method permits the simultaneous visualization of radioactively labeled cells and neuropeptide-producing cells in the same tissue preparation. Autoradiograms are fixed with weak paraformaldehyde solution prior to photographic processing for subsequent immunoperoxidase staining. With this procedure 3H-estradiol is localized in neurophysin I and arginine vasopressin-producing cells of the mouse supraoptic and paraventricular nuclei, neurophysin-producing cells of the guinea pig hypothalamus, and dopamine-β-hydroxylase-containing neurons of the rat lower brainstem; 3H-dihydrotestosterone in pituitary gonadotropes and thyrotropes; and 3H-1,25-(OH)2 vitamin D3 in pituitary thyrotropes and pancreatic B-cells.
Combined Autoradiography and Formaldehyde-induced Fluorescence Methods for Localization of Radioactively Labeled Substances in Relation to Monoamine Neurons
Authors: Grant Lester D. , Stumpf Walter E.
Published: April 2011

A combined technique of formaldehyde-induced fluorescence (FIF) and autoradiography is described for the localization of radioactively labeled substances in relation to monoamine neurons. This method permits the simultaneous visualization of 3H-labeled steroid hormone or drug uptake sites and fluorescing monoamine neural elements (cell bodies, fiber projections, terminals) in the same tissue section. Thin frozen sections cut in a cryostat are freeze-dried, exposed to formaldehyde vapor at 80°C, and carried through dry-mount autoradiography processing steps before fluorescence microscopy screening. Subsequent histological staining of sections and light microscopy are employed for conventional autoradiogram screening. With this procedure, 3H-estradiol and 3H-dihydro-testosterone are localized in various catecholamine (CA) neurons in the diencephalon and lower brain stem of the rat. Also, catecholaminergic as well as noncatechol-aminergic sex steroid target neurons are seen to be innervated by CA terminals in various rat brain regions.
High-Speed Electron Microscope Autoradiographic Studies of Diffusible Compounds,
Authors: Mizuhira Vinci , Shiihashi Michiko , Futaesaku Yutaka
Published: April 2011

Three important factors are necessary for successful electron microscope autoradiography (EM-ARG): good resolution, proper preparation of the radioactive isotope (RI) labeled diffusible compounds, and shortened exposure time for ARG. The resolution problem is fundamental to EM-ARG. However, unless the diffusible RI compounds have been fixed correctly in the tissues during preparation, good resolution is useless. It is also necessary to shorten the exposure time for ARG. As yet, a high-speed ARG method for electron microscopy has not been reported, although scintillation ARG methods have been applied to macro- and micro-ARG since 1960. High specific activity, a large amount of radioactivity per unit exposure for radio incorporation (incubation), and careful selection of labeled compounds that concentrate in the DNA or RNA of cell organelles may increase the sensitivity of the emulsion and shorten the exposure time for ARG. For example, labeled thymidine accumulates in nuclear DNA, 3H-SPG (Schizophyllan-produced polyglucan) is incorporated into lysosomal granules, and labeled iodine concentrates in thyroid follicles, often increasing the sensitivity of the emulsion and shortening the exposure time. High-speed ARG yields good data in a very short time, but high-resolution ARG continues to be necessary, even though it requires 4 weeks or more of exposure time. Scintillation autoradiography using tritium seems unstable. We propose a new way to shorten exposure time for EM-ARG, by combining overdevelopment with coating both sides of the grid with emulsion. This method is approximately 100 times more sensitive than the conventional method, and only 4 days of exposure time are required, in contrast to the 1 month usually needed.
Quantification of in Vivo 3H-Estrogen Uptake by Individual Anterior Pituitary Cell Types of Male Rat:
Authors: Keefer Donald A.
Published: April 2011

A refined version of the combined dry-mount auto-radiographic-immunocytochemical technique (Keefer DA, Stumpf WE, Petrusz P: Cell Tissue Res 166:25, 1976) is described in detail. In vivo nuclear estrogen uptake is measured by silver grain counting in immunocytochemically stained gonadotropes (G), somatotropes (S), lactotropes (L), corticotropes (C), and thyrotropes (T) of male rats. In rats 1 day after orchidectomy and adrenalectomy, the order of nuclear estrogen uptake was S = L G = C T, with T concentrating less than half as much radioactivity as L or S. Fifteen percent of anterior pituitary cells neither concentrated estrogen nor stained immunocytochemically. Estrogen uptake was examined in gonadotropes 1, 14, and 50 days after orchidectomy and was found to be identical at all three times. Estrogen uptake in gonadotropes of rats 14 days following orchidectomy and treatment with progesterone was reduced significantly. Guidelines for reliable quantification of the autoradiographic data is discussed.
Cytochemical hybridization with fluorochrome-labeled RNA. II. Applications.
Authors: Bauman J G , Wiegant J , van Duijn P
Published: April 2011

The cytochemical detection of specific DNA sequences by hybridization with fluorochrome-labeled RNA and detection of the hybrids by fluorescence microscopy is described. RNAs complementary to the DNA of the kinetoplasts of Crithidia luciliae (an insect trypanosome) or to adenovirus-5 (Ad-5) DNA were labeled with the hydrazine derivative of tetramethylrhodamine isothiocyanate (TRITC). The specificity of the reactions between the complementary RNAs labeled both with 3H and tetramethylrhodamine was studied by cross-hybridization experiments using a model system in which the DNAs were bound to Sepharose beads. The extent of the reaction was measured by scintillation counting of the bead suspensions and quantitative fluorescence microscopy of individual Sepharose beads. The ability of the rhodamine-labeled cRNAs to hybridize and the absence of interference of the fluorochrome label with the specificity of the hybridization reaction was thus demonstrated. After cytochemical hybridization on microscopic preparations of C. luciliae cells the rhodamine-labeled kinetoplast cRNA stains only the kinetoplasts. No fluorescence was observed in the nuclei. After cytochemical hybridization of rhodamine-labeled Ad-5 cRNA with virus infected KB cells a distinct staining pattern in the nuclei was observed. No fluorescence was seen in uninfected cells, or after hybridization with heterologous rhodamine-labeled RNA. The possibilities and limitations of cytochemical hybridization with rhodamine-labeled RNA are discussed.