bluuebunny/crossref_metadata_2025 huggingface dataset:
The cytochemical demonstration of GERL in rat hepatocytes during lipoprotein mobilization.
Authors: Novikoff P M , Yam A
Published: April 2011

When a semisynthetic diet containing 1% orotic acid (OA) is fed to rats, the endoplasmic reticulum (ER) of hepatocytes vesiculates and lipoprotein (LP) droplets accumulate within the vesicles. When clofibrate (ethyl chlorophenoxyisobutyrate, CPIB) is added to the orotic acid-rich diet, the ER cisternae reform and the LP is mobilized through the reconstituted ER. A remarkable restoration of normal hepatocyte ultrastructure occurs except for a few organelles. From their morphological appearance it was suggested that cisternae which became dilated with small LP particles were part of GERL, abnormally enlarged. The present communication validates this interpretation through ultrastructural cytochemistry which can distinguish GERL from the adjacent Colgi apparatus. GERL shows acid phosphatase (AcPase) but not thiamine pyrophosphatase (TPPase) activity. In contrast, the adjacent Golgi element shows thiamine pyrophosphatase but not acid phosphatase activity. From such cytochemical studies we have recently proposed that GERL in normal rat hepatocytes may be involved in transforming LP particles, by enzymes like lipases that were presumed to be present in this hydrolase-rich portion of smooth ER. In the situation studied in this communication, the addition of ethyl chlorophenoxyisobutyrate to the diet causes the release from the ER of large amounts of LP to the Golgi apparatus and to GERL. Apparently the capacity of GERL to metabolize LP is exceeded and lipid accumulates in the residual bodies.
Changes in numbers and heparin content of peritoneal fluid mast cells of growing rats measured by flow cytofluorometry.
Authors: Berlin G , Enerbäck L
Published: April 2011

A cytofluorometric method, based on berberine staining of mast cell heparin, was used for flow cytofluorometric counting and heparin quantitation of mast cells in crude peritoneal suspensions of growing rats. The automatic flow cytofluorometric counting of mast cells correlated well with hemocytometer cell counts. The mean mast cell heparin content obtained by flow cytofluorometry showed good agreement with such obtained by cytofluorometry of microscopically identified mast cells. The number of peritoneal mast cells and the mean mast cell heparin content was found to increase as the animals grew older. The results of the microscope fluorometric measurements suggested that the heparin content was normally distributed within mast cell populations of both young and old rats. However, the heparin distributions obtained by flow cytofluorometry were often positively skewed but did not fulfill the condition of the log-normal distribution.
The cytochemical demonstration of beta-glucuronidase in colon neoplasms of rats exposed to azoxymethane.
Authors: Brown C A
Published: April 2011

Colon tumors induced with azoxymethane in male Fischer rats were cytochemically analyzed for beta-glucuronidase using naphthol AS-B1 glucuronide (6-bromo-2-hydroxy-3-naphthoyl-O-anisidine) as a substrate and hexazonium pararosanin as a diazo reagent. This method effectively localizes the bulk of beta-glucuronidase in the surface epithelium, the lamina propria and in the endothelial cells of the lymphoid sinuses and postcapillary venules. Polypoid lesions, adenocarcinomas and mucinous adenocarcinomas show no difference in the amount or in the localization of beta-glucuronidase; however, mucinous adenocarcinomas show a slight increase in the amount of beta-glucuronidase. The few tumors that did metastasize to lymph nodes did not show any difference in their enzyme patterns. Intestinal crypts that show a change in size and shape have a definite increase in beta-glucuronidase activity. An increase in the activity of this enzyme can also be seen in well defined neoplasms as opposed to normal areas of the colon.
Glycosyltransferases in plant and animal tissues effects of fixation and lead on enzyme activity.
Authors: Klohs W D , Goff C W , Bernacki R J
Published: April 2011

As the initial step toward the cytochemical localization of glycosyl-transferases in situ, biochemical determinations of these enzyme activities from onion root tips and L1210 cells were performed before and after fixation as well as in the presence of lead ions. Glycosyltransferase activity from roots fixed in buffered formaldehyde or glutaraldehyde before homogenization decreased as the concentration of the fixative or fixation time was increased. Formaldehyde fixation was less inhibitory than glutaraldehyde; 35% of the glycosyltransferase activity was retained after 30 min fixation in 2% formaldehyde while 25% of the enzyme activity remained after a similar fixation in glutaraldehyde. Substantially higher levels of L1210 cell glycosyltransferase activity were retained after a 30 min 2% formaldehyde fixation (60% sialyltransferase; 82% galactosyltransferase), but inhibition by glutaraldehyde was similar to that observed for onion root galactosyltransferase. Glycosyltransferase from formaldehyde-fixed roots was inhbited 35% by lead nitrate, but sialytransferase from formaldehyde-fixed L1210 cells was unaffected by lead ions. These findings are encouraging for further studies aimed at the development of cytochemical technique to localize glycosyltransferase in plant and animal tissues.
Multiple forms with glucose 6-phosphate dehydrogenase activity in Musca domestica L. as revealed by electrophoresis on cellulose acetate gel.
Authors: Cima L , Malacrida A , Gasperi G , Sacchi L , Grigolo A
Published: April 2011

Single newly emerged males of Musca domestica, WHO strain, usually show five electrophoretic bands of glucose 6-phosphate dehydrogenase (G6PD) activity. Of these five molecular forms, designated with Roman numerals in order from the origin, we have considered the first three: these have been characterized with respect to their substrate and coenzyme specificity and to their sensitivity to some sulfhydryl inhibitors. The data show band III to be G6P specific, nicotinamide adenine dinucleotide phosphate dependent and to be a type I enzyme according to Kamada and Hori's classification. Bands I and II, on the other hand, show wide substrate specificity and low sensitivity to the sulfhydryl inhibitors assayed. In addition, in the absence of an exogenous substrate and in the presence of nicotinamide adenine dinucleotide as a coenzyme, fairly weak bands, which can be ascribed to the so called "nothing dehydrogenase" effect, are seen in the position I and II. Nevertheless, the data reported do not allow a clear definition of the enzymatic type corresponding to bands I and II of G6PD activity.
The in vitro conversion of a specific molecular form of glucose 6-phosphate dehydrogenase from Musca domestica L.
Authors: Gasperi G , Malacrida A , Cima L , Sacchi L , Grigolo A
Published: April 2011

A nicotinamide adenine dinucleotide phosphate dependent glucose 6-phosphate dehydrogenase (G6PD), belonging to type I of Kamada and Hori's classification, is present on the zymograms of newly emerged males of Musca domestica. It is capable of undergoing tryptic degradation and being thus transformed into a different active enzymatic form, with some of its catalytic properties unchanged, but with different electrophoretic mobility. We show in this paper that this specific G6PD form of gut origin in M. domestica is not a tissue-specific enzyme, but rather a product of hydrolytic degradation by gut proteinases which act during the process of homogenization. Besides, the G6PD of type I in the housefly is shown to be sensitive to the "storage effect" and to protection by mercaptoethanol, contrary to its hydrolytic gut form which is not sensitive to these processes. In this connection, we discuss the possible reasons for these differences in behavior.
Localization of chitin in algal and fungal cell walls by light and electron microscopy.
Authors: Pearlmutter N L , Lembi C A
Published: April 2011

Chitin was visualized in cell walls after hydrolysis with potassium hydroxide and subsequent postfixation of the deacetylated polysaccharide (chitosan) in OsO4. Areas of chitin deposition appeared dark borwn by light microscopy and electron dense in the electron microscope. With this method, the presence of chitin was demonstrated in the cell walls of the green alga Pithophora oedogonia (Montagne) Wittrock and two fungi, Ceratocystis ulmi Buism. (C. Moreau) and Blastocladiella emersonii Cantino and Hyatt. Most of the chitin in P. oedogonia ws found in the crosswall disk and small amounts occurred in the outer longitudinal walls. The septal disk of C. ulmi also contained chitin, but significant amounts were present in the inner and outer regions of longitudinal walls as well. Chitin was present throughout the walls of B. emersonii. Small amounts of chitin were not easily demonstrated by this technique, but removal of chitosan by exposure to dilute acetic acid before osmium fixation disrupted cell wall integrity, suggesting that small amounts of the structural polysaccharide had been removed.
The use of epon embedding with the Falck-Hillarp technique to preserve the serotonin-associated fluorescence of endocrine and mast cells in the rat stomach.
Authors: Rubin W , Cosio L
Published: April 2011

Epon embedding was used with the Falck-Hillarp Technique in a study of rat gastric endocrine cells in order to improve morphological resolution. The epon embedding not only produced excellent morphological results, but also resulted in the long-term preservation, now for 10 years of the serotonin-associated fluorescence in the gastric endocrine and mast cells.
Permanent fluorescent staining of nucleic acids in isolated cells.
Authors: Malinin G I
Published: April 2011

Staining of fixed cells, blood smears and chromosomes with 0.1% w/v of 3, 5, 7, 2', 4'-pentahydroxyflavanol (morin dehydrate) in 70% ethanol after brief mordanting with 5% w/v aluminum ammonium sulfate results in permanent fluorescence of cellular nucleic acids. Incubation in ribonuclease solution before mordanting, or 5-min hydrolysis with INHCL at 60 degrees C selectively abolishes RNA fluorescence, while the incubation in deoxyribonuclease solution abolishes DNA fluorescence. The morin-mordant complex bound to nucleic acids is stable to photodegradation and up to five years of storage.
Lactate-dehydrogenase activity in the rat testis: a comparison between fluorometric assay of freeze-dried sections and histochemical localization with phenazine methosulphate.
Authors: Heywood L , Blackshaw A
Published: April 2011

The activity of lactate dehydrogenase (LDH) in freeze-dried sections of rat testes was determined by using a fluorometric assay method and found to be 4.47 +/- 0.23 moles/Kg dry weight/hr (MKDH +/- S.E.M.) in whole sections, 3.31 +/- 0.16 in tubules and 12.0 +/- 1.9 in interstitial tissue. The activities and regional variation are similar to those measured in nervous tissue and are well correlated with the histochemical localization of LDH activity when phenazine methosulphate (PMS) is not used as an electron carrier. LDH and lipoamide dehydrogenase activity have the same histochemical distribution and there is no nonspecific staining with either method. The use of PMS results in reduced dependence on substrate and coenzyme and does not indicate higher interstitial activity but may provide an indication of developing lactate metabolism in maturing sperm. It is recomended that methods with and without PMS be used in studies of LDH activity in the testis.
Rapid hypotonic method for flow cytofluorometry of monolayer cell cultures. Some pitfalls in staining and data analysis.
Authors: Fried J , Perez A G , Clarkson B D
Published: April 2011

The rapid hypotonic staining procedure developed by Krishan for DNA determinations by flow cytofluorometry has been proven accurate for in vivo cell samples and for cell lines growing in suspension culture. We show that the unmodified procedure may produce distorted DNA histograms when used for staining cells growing in monolayer cultures, however. To eliminate these distortions, it was necessary to avoid the use of trypsin by staining the attached cells directly, using a hypotonic fluorochrome solution to which nonionic detergent was added. Two sublines of HeLa S3 cells are shown to exhibit major differences in their staining characteristics. By using our revised staining procedure, the two sublines appear to produce very satisfactory DNA histograms. However, in only one subline does the S phase fraction calculated from the histograms agree with the autoradiographical labeling index. Mitotic cells remain intact under these staining conditions, and the principal observed effect of nonionic detergents in this case is to decrease the coefficient of variation of fluorescence intensity.
Quantitative introduction of a given macromolecule into cells by fusion with erythrocyte ghosts using a fluorescence activated cell sorter.
Authors: Mekada E , Yamaizumi M , Uchida T , Okada Y
Published: April 2011

FITC-conjugated bovine serum albumin (FITC-BSA) molecules were quantitatively introduced into human erythrocyte ghosts by gradual hemolysis. When the ghosts and L cells were fused with UV-inactivated HVJ (Sendai virus), FITC-BSA was introduced into the cytoplasm of the L cells and fluorescence could be observed inthe cells with a fluorescence microscope. A mixture of L cells and ghosts was introduced into a fluorescence activated cell sorter (FACS), which could separate the mononuclear cells on the basis of their light-scattering profile. Four distinct populations of mononuclear cells were found by fluorescence analysis. These populations were separated from the cell mixture and found to correspond to cells fused with one, two and three ghosts and unfused cells. After separation, the cells from each population could form colonies in culture. As a given macromolecule can be quantitatively introduced into erythrocyte ghosts with the FITC-BSA, after fusion of these ghosts with cells, this sorting method is useful for separating cells containing a definite number of macromolecules.
Immunohistochemical localization of thyroid hormone in rat thyroid gland.
Authors: Wilson M , Hitchcock K R , DeLellis R A
Published: April 2011

Direct and indirect immunofluorescence techniques were used to localize the thyroid hormones triidothyronine (T3) and thyroxine (T4) in adult rat thyroid gland. Optimum dilutions of the antisera were established and four tissue fixatives were investigated for usefulness in this technique. Use of antibodies specific for either T3 or T4 resulted in brilliant fluorescence in the colloid pools and apical cytoplasm of follicular cells. In all cases, the adjacent parathyroid gland was devoid of fluorescence. This report demonstrates that these dipeptide hormones can be localized by using immunofluorescence techniques.
Oxygen incineration and electron microscope x-ray microanalysis of mineral particles in biological tissues.
Authors: Henderson W J , Melville-Jones C , Wilson D W , Griffiths K
Published: April 2011

Techniques employed for the recovery from biological tissues of noncombustible fine particles such as asbestos, talc, kaolin and diatomaceous material were assessed by electron microscope x-ray microanalysis. Recovery procedures which have been proven successful for lung tissue were found to be impracticable for more solid types of tissues. Digestion techniques employing acids, alkalis or enzymes and standard incineration procedures were found to be unsatisfactory for human adrenal, cervix, liver and ovarian tissues when the resultant residues were examined by electron microscope microanalysis. The recovered particles were often completely masked with residues which were shown to be composed of organic elements. The use of oxygen during the incineration process completely removed this contaminating material in nearly all cases studied. When such procedures were used, clearly defined particles were recovered from the tissue, thereby permitting x-ray analysis. A quantitative analysis could then be made to estimate the particle content in a variety of tissues.
The pH of the hamster sperm acrosome.
Authors: Meizel S , Deamer D W
Published: April 2011

The pH of the hamster sperm acrosome was estimated by a method based on the distribution of monoamines between membrane enclosed volumes maintaining pH gradients. A fluorescent amine, 9-aminoacridine, was used to permit both microscopic and fluorometric measurements of amine distribution. Cauda epididymal hamster sperm incubated with 9-aminoacridine accumulated the amine in the acrosomal volume. In the presence of NH4Cl or the ionophore Nigericin (compounds which discharge pH gradients) 9-aminoacridine fluorescence disappeared from the acrosome. Amine distribution between the acrosome and external volume was estimated by fluorometric measurement of sperm filtrates in the presence and absence of NH4Cl and Nigericin. These values, together with an estimated acrosomal volume of 0.4mu3 were used to calculate an acrosomal pH of less than 5. In addition, an acrosomal pH of 5 or less was obtained with 14C-methylamine. We suggest that such an acidic acrosomal pH of 5 or less could serve to inhibit the activation or autoactivation of the acrosomal zymogen proacrosin to acrosin, a trypsin-like enzyme involved in fertilization.
Distribution of gamma-aminobutyric acid, glycine, glutamate and aspartate in the cochlear nucleus of the rat.
Authors: Godfrey D A , Carter J A , Lowry O H , Matschinsky F M
Published: April 2011

The distributions of gamma-aminobutyric acid (GABA), glycine, glutamate and aspartate were measured in cochlear nuclei of two rats by quantitative histochemical mapping procedures. The levels and distributions in the two rats were comparable, and resembled those previously reported for cat cochlear nucleus. The results are consistent with a concept that these putative transmitter amino acids have similar levels and distributions in the cochlear nucleus among mammals.
Measurement of cellular DNA mass by flow microfluorometry with use of a biological internal standard.
Authors: Tannenbaum E , Cassidy M , Alabaster O , Herman C
Published: April 2011

Use of a biological standard (chicken erythrocytes) mixed with experimental cell populations allows control of all variables in flow microfluorometric determination of DNA content. These variables include both staining and instrument procedures. In addition, the use of a biological standard allows determination of cellular DNA mass in unperturbed cell populations. DNA mass measured by FMF technique correlates closely with values reported in the literature that used biochemical techniques.
Tissue fixation and staining with osmium tetroxide: the role of phenolic compounds.
Authors: Nielson A J , Griffith W P
Published: April 2011

It has been postulated that phenol-containing areas of plant and animal tissues were osmiophilic, but proof of direct interaction between osmium tetroxide and phenolic materials, or the nature of such reactions, has been lacking. We find that, under conditions similar to those of normal tissue fixation, osmium tetroxide reacts rapidly with those phenols containing o-dihydroxy groups (including such species found in plant tissues) to give very stable chelate complexes. We conclude that these complexes are responsible for the observed electron-density in phenol-containing areas of tissue treated with osmium tetroxide, so that such phenols are indeed osmiophilic.
An efficient method of antibody elution for the successive or simultaneous localization of two antigens by immunocytochemistry.
Authors: Tramu G , Pillez A , Leonardelli J
Published: April 2011

The method described in this paper is available for removal of antibodies retained by tissue antigen after immunohistochemical staining. Its application to the antehypophysis has allowed the successive or the simultaneous localization of two different hormones.
Localization of kallikrein in the coagulating and submandibular glands of the guinea pig.
Authors: Schachter M , Maranda B , Moriwaki C
Published: April 2011

Antibody to pure kallikrein from the coagulating gland of the guinea pig was used to localize kallikrein in the gland by immunofluorescence techniques. This antibody also reacted with the guinea pig's submandibular gland kallikrein. The specific fluorescence in the coagulating gland was present diffusely in all secretory cells lining the crypts. In contrast to its diffuse location in the coagulating gland, kallikrein in the submandibular gland was specifically located in the luminal border of striated and some larger duct cells, whereas the acinar cells and interstitial tissue showed no significant fluorescence.
Immunohistochemical and ultrastructural distribution of antibodies to troponin-C and troponin-I in normal and dystrophic chicken skeletal muscle.
Authors: Wilson F J , Camiscoli D , Irish M J , Hirabayashi T
Published: April 2011

The pectoral muscles from normal and dystrophic chickens were reacted with rabbit antisera to troponin-C and to troponin-I, and the distribution of antibodies was determined by fluorescence microscopy of antibody-stained myofibrils and immuno-electron microscopy of separated I band segments. Chickens of dystrophic strain 308 and control New Hampshire hens were used in this work. Myofibrils which were prepared from both normal and dystrophic muscles and reacted with anti-troponin-I were fluorescent in the I band and A band regions. The Z lines and H zones were unstained. Myofibrils prepared from normal pectoral muscle and treated with anti-troponin-C yielded a pattern of fluorescence similar to that for anti-troponin-I treated myofibrils. However, those myofibrils isolated from dystrophic muscle and reacted with anti-tropinin-C had a weak fluorescence over their entire lengths, and discrete A- and I-band staining was not visible. These results were confirmed by ultrastructural studies of separated I segments reacted with the antisera. It is concluded that in the dystrophic muscle either the antigenic sites of troponin-C are changed which results in a loss of antibody-combining ability or these sites are masked in some way which prevents the reaction with the antibody.
The cytochemical localization of adenylate cyclase: fact or artifact?
Authors: Kempen H J , de Pont J J , Bonting S L , Stadhouders A M
Published: April 2011

In a study of the location of adenylate cyclase activity in rat pancreas with the method of Reik et al. (Science 168:382, 1970), as modified by Howell and Whitfield (J Histochem Cytochem 20:873, 1972) it was found that (a) unspecific staining occurs in rat pancreatic tissue fragments incubated in the Reik-Howell medium in the absence of substrate; (b) addition of adenylyl-imidodiphosphate (AMP-PNP) as substrate, either alone or together with stimulants of rat pancreas adenylate cyclase (secretin. NaF), does not result in increased precipitation; (c) cytochemical incubation of isolated rat pancreatic acinar cells and of rat liver and kidney fragments does not lead to substrate-specific precipitation. In subsequent chemical studies we have found that cyclic adenosine monophosphate (AMP) formation from [alpha32P]AMP-PNP in the presence of rat pancreatic particulate matter is very low in the Reik-Howell medium without lead ions, but is stimulated by addition of lead nitrate (4 mM). Whereas heat-treatment of the particulate matter abolishes all cyclic AMP formation in the absence of lead ions, it actually increases cyclic AMP production in the presence of 4 mM lead nitrate. This indicates that the cyclic AMP formation in the complet Reik-Howell medium occurs by a nonenzymatic mechanism. In addition, this medium shows a tendency to become turbid, particularly when calcium ions are added to the medium, suggesting a possible explanation for the apparently specific cytochemical detection observed by other authors. A revised cytochemical medium, with barium replacing lead and with a pH of 8.9 (optimal for adenylate cyclase with AMP-PNP substrate), leaves rat pancreatic adenylate cyclase activity intact and hormone sensitive, while it is still able to precipitate imidodiphosphate. However, cytochemical incubation of isolated rat pancreatic acinar cells in this revised medium in the presence of AMP-PNP and secretin does not yield an electron-dense precipitate, showing that the enzyme activity is to low to produce sufficient imidodiphosphate. These findings throw further doubt on the validity of the cytochemical detection of adenylate cyclase, reported by other investigators, notwithstanding the alleged positive results.
Use of immunocytochemical techniques for the localization of human placental lactogen.
Authors: Watkins W B
Published: April 2011

The recent claim by Gau and Chard (Br J Obstet Gynaecol 83:876, 1976) that, on theoretical grounds, it may be impossible to demonstrate the presence of human placental lactogen in placental tissue using the immunoperoxidase technique, has been reinvestigated. Placental tissue fragments fixed in Carnoy's fluid retained their morphologic identity compared with tissue fixed in formalin. Using these nonformalin fixed tissues, human placental lactogen was successfully localized within the cytoplasm of the syncytial layer of the placental villus. It is concluded that placental villi at term do in fact contain sufficient human placental lactogen to be demonstrated using the immunoperoxidase technique in contrast to the observation of Gau and Chard.
Peroxidase-positive endothelial cells in sinusoids of the mouse liver.
Authors: Stöhr G , Deimann W , Fahimi H D
Published: April 2011

The cytochemical localization of endogenous peroxidase activity in sinus lining cells of mouse liver has been investigated. Kupffer cells, as identified by their exclusive ability to phagocytize large (0.8 micron) latex particles, exhibited strong peroxidase activity in nuclear envelope and endoplasmic reticulum. In addition, weak to moderate peroxidase activity was found in 57% of all endothelial cells. The enzyme in endothelial cells was also localized in nuclear envelope and endoplasmic reticulum, with a negative reaction in the Golgi apparatus. These observations indicate that peroxidase staining, as a marker for identification of Kupffer cells in mouse liver, is only of limited value and should be used in conjunction with other methods (e.g., latex phagocytosis).
A new method for the study of glutaraldehyde-induced crosslinking properties in proteins with special reference to the reaction with amino groups.
Authors: Molin S O , Nygren H , Dolonius L
Published: April 2011

A new method for the study of glutaraldehyde reactions with proteins is presented. Glutaraldehyde-reacted protein is in a first step isolated and then in a second step reacted with aminohexyl groups bound to Sepharose particles. This reaction is linear at low protein concentrations and proceeds rapidly when proteins are reacted with 100-fold and 1000-fold molar excess of glutaraldehyde. This method enables the study of glutaraldehyde-induced crosslinking properties of the modified proteins as an isolated property with high reliability.
Cyclic 3',5'-nucleotide phosphodiesterase; cytochemical localization in rat renomedullary interstitial cells.
Authors: Florendo N T , Pitcock J A , Muirhead E E
Published: April 2011

The localization of cyclic 3', 5' -nucleotide phosphodiesterase activity in rat renal papillae was examined by utilizing cytochemical methods. Renal medullary interstitial cells had predictable phosphodiesterase activity predominantly on the cytoplasmic border of dilated cisternal membranes. Cells of the collecting tubule and loop of Henle contained diffuse reaction product. Capillaries had reaction product localized in pinocytic vesicles. Addition of theophylline resulted in no deposition of reaction product in interstitial cells and in cells of the collecting tubule and loop of Henle, suggesting an inhibition of phosphodiesterase activity. Since the membranes of dilated cisternae of renal medullary interstitial cells have been shown to be related to prostaglandin synthesis and probably to the anti-hypertensive function of these cells, the finding of phosphodiesterase activity on these membranes suggests a possible role of cyclic AMP in these two functions.
Rhodamine immunohistofluorescence applied to plant tissue.
Authors: Hapner S J , Hapner K D
Published: April 2011

Tissue slices from the roots and seeds of sanifoin (Onobrychis viciifolia, Scop.) exhibit bright autofluorescence when illuminated with blue (495 nm) light. This autofluorescence is indistinguishable from the fluorescence emission of fluorescein, the commonly used fluorochrome in immunohistochemical staining procedures. Rhodamine isothiocyanate, when coupled to immunoglobulin, and excited with green light at 546 nm, exhibits a reddish-orange fluorescence with an emission maximum at 590 nm. Plant tissue has little or no autofluorescence when illuminated at this wavelength and viewed with a 580 nm barrier filter. Therefore, use of rhodamine for immunohistochemical localization in plant tissue avoids interpretative complications due to inherent autofluorescence.
Localization of carbohydrate components in rat colon with fluoresceinated lectins.
Authors: Essner E , Schreiber J , Griewski R A
Published: April 2011

Cryostat sections of rat descending colon were studied by fluorescence microscopy after exposure to conjugates of fluorescein isothicoyanate with lectins from Glycine max (soybean), Triticum vulgaris (wheat germ), Ricinus communis (castor bean), Ulex europaeus, (gorse), Dolichos biflorus (horse gram) and Canavalia ensiformis (concanavalin A) (Jack bean). No two lectins showed identical patterns of fluorescence. FITC-conjugates of soybean and D. biflorus lectins reacted strongly with the mucus present in the crypt lumens and with the surface (as well as cytoplasm) of the epithelial cells suggesting that these sites are rich in terminal, non-reducing, N-acetylgalactosamine residues. Wheat germ, R. communis, U. europaeus and concanavalin A-FITC conjugates did not stain mucus but showed fluorescence in the cytoplasm of absorptive cells as well as in the lamina propria and submucosa. The FITC-R. communis conjugate also reacted with structures in the apical portion of epithelial cells that may correspond to the Golgi apparatus.
Receptors for luteinizing hormone-releasing hormone.
Authors: Sternberger L A
Published: April 2011

Antisera to luteinizing hormone-releasing hormone (LH-RH) confer on Araldite sections of occasional rat pituitaries moderate immunocytochemical staining to the large secretory granules of gonadotrophs. Treatment of the sections with LH-RH before anti-LH-RH yields strong staining in all animals, irrespective of presence or absence of staining without pretreatment. This enhancement of staining is specific for LH-RH and is a high affinity, saturable reaction. Staining with or without LH-RH pretreatment is absent when anti-LH-RH absorbed with insolubilized LH-RH is used. Staining is inhibited by carboxyterminally-deficient LH-RH, unaffected by aminoterminally deficient LH-RH.
Immunostaining of growth hormone and prolactin in paraffin-embedded and stored or previously stained materials.
Authors: Halmi N S
Published: April 2011

In attempts to evaluate immunocytochemically autopsy and biopsy material previously obtained and processed for conventional histologic staining, we had to resort to immunostaining of tissues embedded years ago or even sections already stained with hematoxylin-eosin or aldehyde thionin-PAS-orange G. Hypophysial growth hormone and prolactin proved remarkably resistant to such prior treatment with regard to their antigenic properties, and could be readily immunostained in tissue embedded in paraffin 3-4 years earlier, and after destaining of sections prepared up to 7 years earlier. The results of such "retrospective" immunocytochemical evaluation of autopsy and biopsy materail is illustrated with the staining of "pregnancy cells" for prolactin in the hypophysis of a woman postpartum, the immunostaining for prolactin in the cells of adenomas associated with marked hyperprolactinemia, the staining for growth hormone in adenomas removed from children with gigantism, and the immunostaining for prolactin, growth hormone or both in several adenomas that were discovered at autopsy and not associated with a known clinical history of endocrine aberrations.
Chromium concentration by proximal renal tubule cells: an ultrastructural, microanalytical and cytochemical study.
Authors: Berry J P , Hourdry J , Galle P , Lagrue G
Published: April 2011

After treating rats with potassium bichromate, x-ray microanalysis of the proximal renal tubule cells demonstrated the presence of chromium in intracellular vacuoles. The lysosomal nature of these vacuoles is shown by the visualization of acid phosphatase activity. Chromium is concentrated both in the autolytic vacuoles and in their residual forms. The metal is eliminated quite late, concomitant with cellular necrosis.
The use of polyethyleneimine for demonstration of anionic sites in basement membranes and collagen fibrils.
Authors: Schurer J W , Kalicharan D , Hoedemaeker P J , Molenaar I
Published: April 2011

Strong cationic polyethyleneimine (PEI) is used to trace anionic sites in lung, choroid plexus, glomeruli, blood vessels and also on collagen fibrils. In the mentioned tissues, PEI is found in the basement membranes with a spacing identical to that found on collagen fibrils. It is assumed that the acid endgroups of tropocollagen molecules are responsible for the binding of PEI.
An evaluation of Coomassie Brilliant Blue as a stain for quantitative microdensitometry of protein in section.
Authors: Cawood A H , Potter U , Dickinson H G
Published: April 2011

The specificity and stoichiometry of the binding of Coomassie Brilliant Blue (CBB) to protein in section has been examined using both frozen protein matrices and plant material. The maximum adsorbance of the stain, bound and in solution, was found to be 620 nm although variation in the results at this wavelength necessitated measurements to be made at 600 nm. After enzyme treatments of sectioned plant material embedded in resin, all CBB-binding biological material was shown to be sensitive to non-specific protease. The relationship between optical density at 600 nm and section thickness was tested statistically against the Lambert-Beer law, using microdensitometry of cryostat-sectioned, frozen genatine solution. The analyses showed conclusively that, under these conditions, CBB adheres strongly to the Lambert-Beer relationship. CBB may thus be considered as a very specific protein stain, eminently suited both to cytological observation and quantitative microdensitometry.
Acid lipase: a histochemical and biochemical study using triton X100-naphtyl palmitate micelles.
Authors: Schaffner T , Elner V M , Bauer M , Wissler R W
Published: April 2011

Hydrolsis of a-naphtyl palmitate dispersed with the detergent Triton X-100 at acid pH was studied by a histochemical diazocoupling technique in both fixed sections and cultures of primate tissues as well as by a biochemical assay employing the same chromogenic substrate. Evidence for the exclusive hydrolysis of this artificial fatty acid ester substrate by acid lipases was gathered from (1) comparison of isoelectric focusing zymograms developed with different substrates, (2) kinetic analysis of enzyme activity in the presence or absence of inhibitors, including a natural substrate of acid lipase, trioleylglycerol, (3) specific localization of marked enzyme activity in certain tissues, and (4) absence of detectable enzyme activity in a case of human acid lipase deficiency (Wolman's disease). Histochemically, acid lipase activity was most readily detected in cells active in the uptake and processing of neutral lipids, i.e., the phagocytes of the reticuloendothelial system, the adrenal cortex and the lipid-storing cells in the athero-sclerotic plaques of arteries.
On testing the activity of proteases from human polymorphonuclear neutrophils on blood smears.
Authors: Klessen C
Published: April 2011

A cytochemical method is presented for the demonstration of proteases in human polymorphonuclear (PMN) neutrophils on fixed blood smears. This new technique is based on solubilization of proteases from PMN neutrophils by incubation with 0.25 M NaCl in borate buffer at pH 8.5 which leads to degradation of erythrocytes and plasma in a disclike zone (halo) around centrally situated PMN neutrophils, an effect that is visualized by staining smears using a modified colloidal iron reaction. Halo formation is inhibited by trypsin inhibitors of soya-bean as well as of chicken egg white mucoid and by phenylmethyl-sulfonylfluoride.
Quantation by flow microfluorometry of total cellular DNA in Acanthamoeba.
Authors: Coulson P B , Tyndall R
Published: April 2011

The DNA content of five species of Acanthamoeba was determined by flow microfluorometry. Acanthamoeba castellanii (AC-30), acanthamoeba polyphaga (APG and P-23), acanthamoeba rhysodes, acanthamoeba culbertsoni (A-1), and acanthamoeba royreba were grown in a casitone based medium 24-48 HR. The trophozoites were harvested, and evaluated for DNA-bound fluorescence. All species tested has DNA values between 2.0-5.0 pg/cell. These results placed DNA/cell values of Acanthamoeba slightly lower than DNA/cell values of other eucaryotic cells and much lower than Amoeba proteus values. These results indicate that FMF may be a useful adjunct in distinguishing Acanthamoeba cells from either eucaryotic cells or some other amoeba. However, differences in DNA/cell between species of Acanthamoeba are small and would not be useful in identification of species.
Ultrastructural localization of acetylcholinesterase in cultured cells. III. DFP treated embryo muscle.
Authors: Golder T K , Nieberg P S , Wilson B W
Published: April 2011

Brief treatment with 10(-4)M diisopropylfluorophosphate (DEP) irreversibly inactivates acetylcholinesterase (E.C.3.1.1.7; acetylcholine hydrolase) (AChE) activity in 10 day old chick embryonic muscle cultures. Electron microscopic cytochemistry was employed to follow the distribution of new AChE during recovery from DEP treatment. In normal 10 day cultures of embryo pectoralis muscles AChE is localized in the nuclear envelope, perinuclear sarcoplasm, sarcotubular system, subsurface vesicles and bound outside the cells. Immediately after DFP treatment AChE activity is absent in large myotubes. Within 15 min, activity is randomly present in small amounts in the sarcotubular system and nuclear envelope. There is a dramatic increase in activitv in the nuclear envelope during the 1st hr of recovery, and connections between the nuclear envelope and sarcotubular system are often seen. The next few hr of recovery show increased AChE activity. By 4 hr activity approaches that of controls. Six to 8 hr after treatment, AChE activity can be detected spectrophotometrically in the medium and can be seen bound outside the cells with the electron microscope. The spatial and temporal patterns of AChE activity demonstrate that the recovery of AChE and its mobilization and release from DFP-treated cells are not governed solely by the levels attained by the enzyme in the cultured embryo muscle.
Ultrastructural localization of nonheme celluar iron with ferrocyanide.
Authors: Parmley R T , Spicer S S , Alvarez C J
Published: April 2011

The Prussian blue reaction was evaluated at the ultrastructural level as a cytochemical method to identify ferric and ferrous iron in rat bone marrow and splenic macrophages. Satisfactory tissue preservation and staining were achieved after fixation for 1 hr in 3% glutaraldehyde and exposure for 30 min to Perls's ferrocyanide solution before routine osmication and embedding. The acid ferrocyanide solution formed cuboidal and irregular electron-opaque deposits which localized ferric iron in the macrophage siderosomes and hyaloplasm. When thin sections were directly stained with the acid ferrocyanide, the stain deposits were much less distinct. The size and number of cytes exhibited sparse evenly distributed stain deposits. Several cells displayed abundant precipitates on the inner surface of the plasmalemma. Prussian blue precipitates were occasionally seen in mitochondria and nuclear euchromatin. Although osmium tetroxide post-fixation improved tissue preservation, it did not enhance the density of the ferri-ferrocyanide precipitate. The ferrocyanide solution yielded cuboidal deposits also in clots impregnated with ferritin, and electron diffraction analysis confirmed the symmetrical crystal structure of these stain precipitates. Smaller irregular precipitates were formed in clots impregnated with FeCl3, or Fe2 (SO4)3 solutions, despite the equally interpreted as indicating that the iron hydroxide core or protein structure of ferritin and hemosiderin contributed to the formation of the ultrastructurally evident cuboidal precipitates, but were not necessary for the formation of a colored reaction product. The acid ferrocyanide solution failed to stain clots formed in FeCI2, CuCI2 or CuCI solutions. Staining with a ferricyanide solution identified only sparse foci of ferrous iron in some siderosomes. This study demonstrates that the Prussian blue reaction can be used ultrastructurally to localize iron cations bound to some nonheme iron binding proteins.
The alpha-glucan-uridine diphosphate glucosyltransferase reation for the identification of glycogen-depleted muscle fibers.
Authors: Pool C W , Donselaar Y E , Griep P A
Published: April 2011

This paper decribes the use of the alpha-glucan uridine di-phosphate glucosyl transferase reaction for enhancing the contrast between glycogen depleted and non-depleted muscle fibers in the periodic acid schiff (PAS) reaction. Muscle fiber glycogen was depleted by prolonged repetitive stimulation of single motor units of the extensor digitorum longus muscle from the rat.
Inferences on RNA synthesis rates during mitosis from flow cytofluorimetric RNA histograms.
Authors: Watson J V , Chambers S H
Published: April 2011

Flow cytofluorimetric techniques with acridine orange staining have been used to study RNA levels of cells during and immediately after mitosis. It can be inferred from the histogram data that, of the mitotic cells, those in telophase have the highest RNA content, and that the rate of synthesis increases rapidly between anaphase and telophase. These inferences correlate with results obtained from parallel labeled precursor studies.
Multiangle light scattering flow photometry of cultured human fibroblasts: comparison of normal cells with a mutant line containing cytoplasmic inclusions.
Authors: Schafer I A , Jamieson A M , Petrelli M , Price B J , Salzman G C
Published: April 2011

Multi-angle light scattering flow photometry was used to study the light scattering properties of normal cultured fibroblasts and a mutant fibroblast line containing cytoplasmic lysosomal inclusions. The effect of glutaraldehyde fixation on the light scattering properties of the cells was also examined and correlated with their ultrastructure. Normal fibroblasts showed uniform organelle distribution with few vacuoles or dense bodies in the cytoplasm while the mutant line showed abnormal cytoplasmic inclusions of varying morphology, density and lucency. As predicted by light scattering theory, the mutant cells containing the cytoplasmic inclusions scattered more light at large angles (greater than theta = 1.85 degrees) than did the normal cells. Glutaraldehyde fixation decreased light scattering at small angles (less than theta = 1.85 degrees), increased light scattering at larger angles (greater than theta = 1.85 degrees) in both normal and mutant cells and enhanced resolution of the light scattering signatures. The mutant line scattered 2-3 times more light at a wide angle (greater than theta = 12.74 degrees) than did the normal cells. These data suggest that abnormal lysosomal storage inclusion bodies in the cytoplasm of the cells can be detected by differential light scattering methods.
Naphthol AS-BI (7-bromo-3-hydroxy-2-naphtho-o-anisidine) phosphatase and naphthol AS-BI beta-D-glucuronidase in Chinese hamster ovary cells: biochemical and flow cytometric studies.
Authors: Dolbeare F A , Phares W
Published: April 2011

Conditions for the biochemical and flow cytometric assay of 7-bromo-3-hydroxy-2-naphtho-o-anisidine phosphatase and beta-D-glucuronidase activities in Chinese hamster ovary cells were studied. In the biochemical assay, the pH optimum for the phosphatase activity was pH 4.6 with a Km of 10(-5) M; the pH optimum for beta-D-glucuronidase activity was pH 5.0 with a Km of 2 x 10(-5) M. For intact cells the derived constants were 3 to 10 times higher. The rate of hydrolysis of both substrates was also examined by flow cytometry. Cellular fluorescence increased linearly for only about 15 min. Diffusion of the fluorescent product probably caused nonlinearity of the fluorescence increase and was demonstrated by mixing cells incubated with substrate with those that had not been incubated. After 15 min, cells that had not been exposed previously to product or substrate contained the fluorescent product. Cells fractionated into size classes by centrifugal elutriation also were analyzed by flow cytometry for beta-D-glucuronidase activity. The activity increased linearly with the increase in cell size corresponding to the progression from G1 through S and into G2-M phases of the cell cycle.
Intracellular pH-determination by fluorescence measurements.
Authors: Visser J W , Jongeling A A , Tanke H J
Published: April 2011

A method was developed to determine the intracellular pH (pHi) of individual cells by use of fluorescence measurements. The method is based on the observation that the fluorescence excitation spectrum of fluorescein is pH-dependent. Fluorescence excitation spectra from individual rat bone marrow cells treated with fluorescein diacetate (FDA) were compared with those of fluorescein solutions of known pH values. Cells which were suspended in media of pH between 4.0 and 8.1 with high to normal buffering capacities had pHi values equal to those of the media. Cells suspended in media with low buffering capacities maintained a pH,i of 6.7 +/- 0.2. Preliminary results indicated that the pHi of individual cells may also be determined by using flow cytometry.
An automated microscope for quantitative cytology combining television image analysis and stage scanning microphotometry.
Authors: Ploem J S , Verwoerd N , Bonnet J , Koper G
Published: April 2011

This paper describes an automated microscope developed for operation in conjunction with the Leyden Television Analysis System. It features automated control of magnification, illumination, movement of scanning stages, and fine focus. These functions are controlled by means of a microcomputer. This enables a flexible design and relieves the supervising computer of simple but time consuming tasks. The combination of an automated microscope and Leyden Television Analysis System provides a powerful tool in quantitative cytological research. The flexible design permits other microscopic functions to be added with relatively little effort.
A double scanning microphotometer for image analysis: hardware, software and biomedical applications.
Authors: Brugal G , Garbay C , Giroud F , Adelh D
Published: April 2011

A new image processing system designed for densitometry and pattern analysis of microscopic specimens is described with special regard to the hardware, the software and the biologic applications. The data acquisition procedure involves the combination between the scanning of the preparation by means of a motorized stage and the scanning of successive fields by a mechanical device. The signal provided by the photomultiplier is converted into digital values which are directed to an on-line computer. The data processing is based on a one-pass computation involving automata theory and therefore it avoids the storage of the image in the computer memory. In so doing, an entire and continuous image of the whole preparation can be processed at the highest magnification of the microscope whatever the size of the analyzed specimen may be. A biologic application of the system is reported and concerns the automatic identification and counting of cells in the various phases of the mitotic cycle.