Stripping Film Research Articles

Assimilation of 14CO 2 by Fusarium oxysporum grown under oligotrophic conditions

Fusarium oxysporum, recently isolated from soil, was grown under strict oligotrophic conditions in the presence of 14CO2. Stripping film autoradiography and acid-washing of mycelium demonstrated the incorporation of the 14C label into the mycelium. Assimilation of 14CO2 continued to occur when the growth environment was purged of volatile organics and carbon monoxide, and partly purged of hydrogen. Killed mycelium did not incorporate 14C, demonstrating an active assimilation. Partitioning of cell components showed most of the assimilated 14C to be present in the wall/wall-associated membranes and a lack of RuBisCo activity in mycelial extract confirmed that CO2 assimilation did not involve the Calvin cycle.

An autoradiographic technique for en-face preparations of aortic endothelium.

A method for the en-face preparation of aortic endothelium was developed from various different published accounts of the technique. This was adapted for autoradiography. Eighteen mice had tritiated thymidine (1 microCi/g body weight) injected and were killed 1 day later. Perfusion fixed aortae were cut into segments and placed face down on gelatin-coated slides. The arterial media and adventitia were removed and the en-face preparations coated with autoradiographic stripping film. After an exposure of 12 weeks the autoradiographs were developed and stained with Harris' haematoxylin. Labelled endothelial cells were clearly distinguishable and there was no distortion of the cells compared with other en-face and whole mount preparations. The technique described is simple, reliable and produces consistent results.

Cerebral protein synthesis during long-term recovery from severe hypoglycemia.

Regional protein synthesis was investigated in the rat brain during long-term recovery from insulin-induced hypoglycemia with 30 min of cerebral electrical silence. At various time intervals up to 14 days after glucose replenishment, animals received a single dose of L-[3,5-3H]tyrosine and were killed 30 min later. Brains were processed for autoradiography using the stripping film technique. Although hypoglycemia sufficiently severe to cause cessation of EEG activity leads to almost complete inhibition of amino acid incorporation in all "vulnerable" forebrain structures (cerebral cortex, hippocampus, caudoputamen), autoradiographs revealed a very specialized sequence with differential posthypoglycemic restoration of biosynthetic activity in certain neuronal cell types. Three major subpopulations could be distinguished: Neurons that fully regained their protein synthetic capacity within 6 h following hypoglycemia (cortical neurons of layer III-VI, large neurons in the caudoputamen, CA3 and CA4 pyramidal neurons, the majority of granule cells of the dentate gyrus) seemed to escape neuronal necrosis. Prolonged impairment of protein synthesis with only partial restoration during the early posthypoglycemic recovery period (CA1 neurons, most small- to medium-sized neurons of the caudoputamen) carried an increased risk of permanent cell damage. The large majority of these neurons, however, showed full recovery of protein synthesis as late as 7 days after hypoglycemia. Neurons with complete lack of amino acid incorporation after 6 h of recovery (granule cells at the crest of the dentate gyrus, small neurons of the dorsolateral caudoputamen) never resumed protein synthesis, regressed, and died. These studies in conjunction with morphological analysis indicate that the sequential recovery of protein synthesis reflects the extent to which neuronal populations are at risk during severe hypoglycemia.

Persistent inhibition of protein synthesis precedes delayed neuronal death in postischemic gerbil hippocampus.

Regional cerebral protein synthesis was investigated in the Mongolian gerbil during recovery from forebrain ischemia produced by bilateral common carotid artery occlusion for 5 min. At various recirculation periods up to 72 h animals received a single dose of L-(3,5-3H)tyrosine and were killed 30 min later. Brains were processed for autoradiography using the stripping film technique. During the initial 30 min of recirculation autoradiographs revealed an almost complete inhibition of protein synthesis in all forebrain structures with the exception of the medio-dorsal thalamic nuclei. Between 30 min and 12 h of recirculation amino acid incorporation was completely restored in neurons of the cerebral cortex, basal ganglia, hippocampal CA3 and CA4 zones and the dentate gyrus. In CA1, early (90-min postischemia) and progressive recovery of a few irregularly dispersed neurons was observed, but the vast majority of pyramidal cells never regained their normal biosynthetic activity. Between 3 and 6 h of recirculation CA1 neurons showed faint labeling, followed by a secondary deterioration resulting in complete lack of incorporation within 12 h after restoration of blood flow. Autoradiographs at all subsequent time points exhibited persistent inhibition of protein synthesis in CA1 until neuronal necrosis occurred 2-3 days later. Thus, in contrast to ischemia-resistant cell populations with rapid progressive and complete restoration of protein synthesis, hippocampal neurons undergoing delayed necrosis are characterized by an early incomplete recovery immediately followed by a secondary persistent inhibition.

Nb 95 and Ni 63 diffusion along the α¦β interphase boundaries of A Zr-2.5 wt% Nb alloy

The diffusion of Nb 95 and Ni 63 has been studied in a Zr-2.5 wt% Nb alloy. The specimens were prepared by means of heat treatments that provided a stabilized two-phase structure, resolvable by optical microscopy, and with natural interphase boundaries. Diffusion of Nb 95 was studied in the 702–818°C temperature range, corresponding to the α + β field. The interphase boundary diffusion coefficients obtained in this work were 3 to 4 orders of magnitude higher than the bulk Nb diffusion coefficient in pure Zr. The Arrhenius plot appeared not quite linear. The activation energy varied from 105 kJ/mol for low temperatures to 230 kJ/mol for high temperatures. Diffusion of Ni 63 was studied at 801 °C by means of autoradiographs. X-ray and stripping film autoradiographs were obtained at several depths, showing at small depths a non uniform tracer distribution, in coincidence with the distribution of both phases. At greater depths the network of interphase boundaries is observed as darker lines.

125I-lysergic acid diethylamide binds to a novel serotonergic site on rat choroid plexus epithelial cells

125I-Lysergic acid diethylamide (125I-LSD) binds with high affinity to serotonergic sites on rat choroid plexus. These sites were localized to choroid plexus epithelial cells by use of a novel high resolution stripping film technique for light microscopic autoradiography. In membrane preparations from rat choroid plexus, the serotonergic site density was 3100 fmol/mg of protein, which is 10-fold higher than the density of any other serotonergic site in brain homogenates. The choroid plexus site exhibits a novel pharmacology that does not match the properties of 5-hydroxytryptamine-1a (5-HT1a), 5-HT1b, or 5-HT2 serotonergic sites. 125I-LSD binding to the choroid plexus site is potently inhibited by mianserin, serotonin, and (+)-LSD. Other serotonergic, dopaminergic, and adrenergic agonists and antagonists exhibit moderate to weak affinities for this site. The rat choroid plexus 125I-LSD binding site appears to represent a new type of serotonergic site which is located on non-neuronal cells in this tissue.

Propidium iodide staining of cytoautoradiographic preparations for the simultaneous determination of DNA content and grain count

A new method is described for staining cell nuclei with propidium iodide in preparations that have been processed for autoradiography. The permeability of the stripping film to the dye molecule has been studied, as have the staining conditions, in order to optimize the stoichiometry of the DNA-dye interaction. A procedure has also been set up to allow the simultaneous measurement of DNA content and grain count on the same cell.

Autoradiography of 32P and 14C incorporation into protoplasts as a means of determining the percentage of virus infected protoplasts

Stripping Film Autoradiography (SFA) was used to measure percentage protoplast infection. Control and infected protoplasts were incubated in the presence of [ 32P]orthophosphate or [ 14C]leucine fixed onto glass slides and placed in contact with a sensitive photographic emulsion. Infected protoplasts were distinguished by blackened areas of exposed film which coincided with protoplasts containing labelled virus products. The use of SFA is demonstrated for two different virus/protoplast systems, namely tobacco mosaic virus (TMV) in tobacco and turnip yellow mosaic virus (TYMV) in rape. The method detects infection as early as 12–16 h post-inoculation and UV irradiation is not necessary for reducing background interference from labelled host proteins.

3H-thymidin einbau in die rattenmilz sowie in die lymphozyten des milzarterien-bzw. milzvenenblutes and des kapillarblutes nach ductus thoracicus blockade

Introduction. After thoracic duct blockade prior to its entry into the left venous angle, there is a fall in the lymphocyte count in the peripheral blood, with an initial minimum after 29 hours. At 32 hours, the lymphocytes rise steeply again despite continued lymph stasis. The question arises as to the origin of these lymphocytes. Since in the rat the spleen is the main site for the influx and efflux of recirculating lymphocytes, the objective of the present studies was to investigate the incorporation of 3H-TdR into lymphocytes of the peripheral blood and spleen after thoracic duct blockade. Materials and Methods. For this purpose, we blockaded the thoracic duct prior to its entry into the left venous angle in adult male and female albino rats. Thirty healthy male or female albino rats were sham operated. Nine healthy male or female animals served as controls. We took daily blood samples from the capillaries of the tip of the tail as well as from the splenic artery and the splenic vein of all animals by means of a leukocyte pipette after one to eight days, and after 10 and 12 days. From these samples, we determined the hematocrit and the lymphocyte content (in per 1000 or per µl). The animals received an intraperitoneal pulse labeling with 3H-TdR (1.0–1.5 µCi/g body weight). On the given experimental days, the animals were killed by decapitation under light ether anesthesia and completely autopsied. In NTB-layered blood smears, we determined the labeling indices (in per 1000 or per µ1) and the labeling intensity of lymphocytes. Furthermore, we determined the arteriovenous difference of the labeled lymphocytes and the percentage distribution of silver grain densities. In the autoradiograms of the spleen coated with stripping film, we determined the labeling indices and the labeling intensity in the periarteriolar lymph sheath, in the follicle center, in the periphery of the perifollicular zone, in the marginal zone, and in the red pulp. From these values, we further determined the arteriovenous difference of labeled lymphocytes in the blood and the percentage distribution of silver grain densities. Results. 1. Lymphocyte content is higher in splenic venous blood than in splenic arterial blood or in capillary blood. 2. After thoracic duct blockade, there is a steep fall in non-labeled lymphocytes with an initial minimum after 29 hours and a second minimum on the fifth postoperative day. 3. The labeling indices of blood lymphocytes show an inversely proportional behavior with an initial maximum after 32 hours and a second maximum after five days. 4. The curve of the arteriovenous differences of labeled lymphocytes shows a first labeling maximum after thirty two hours and a second, smaller labeling maximum on the fifth postoperative day. On the seventh postoperative day, the arteriovenous differences of labeled lymphocytes fell below the initial values, since increased numbers of labeled lymphocytes flow into the spleen. 5. In the determination of the labeling intensity, increased numbers of intensely labeled lymphocytes are demonstrated in the peripheral blood after the second postoperative day. 6. The 3H-TdR labeling indices of the individual spleen zones show a biphasic curve course with a first, smaller narrowly based peak 48 hours after operation and a second higher, broadly based peak between the fourth and the eighth postoperative day. 7. In the determination of labeling intensity, increased numbers of intensely labeled cells are found in all spleen zones after the second postoperative day. 8. The labeling indices of the sham-operated animals attain a labeling maximum on the eighth postoperative day in the periphery of the perifollicular zone and in the red pulp. No significant alteration in labeling indices is found in the T zone during the entire period of the experiment. Eight days after thoracic duct blockade, there is a prolongation of T S, T G2, and part of the M phase. Discussion. The results suggest that as an immediate reaction, the spleen endeavors to compensate for the abrupt fall in the lymphocyte count in the peripheral blood. The final return to normal takes place after eight to ten days with participation of the extralienal lymphatic apparatus.

Uptake of (3H)‐ADTN into dopaminergic neurons in the rabbit retina

6,7‐ADTN (2‐amino‐6,7‐dihydroxytetrahydronaphthalene) has been introduced as a potent dopaminergic agonist (McDermed, McKenzie & Phillips 1975, Cannon et al. 1977) which could possibly be used as a false neurotransmitter for dopamine. However, direct morphological evidence of the cellular localization of (3H)‐ADTN is lacking. This can readily be obtained in the retina where the dopaminergic neurons are well recognizable. We have therefore injected 20 μCi (3H)‐ADTN (2‐(5,8‐3H)‐ amino ‐6,7‐dihydroxy‐1,2,3,4‐ tetrahydronaphthalene, specific activity 35.5 Ci/mol, New England Nuclear, Dreieichenhain, West Germany) into rabbit eyes. After 4 h, the retina was obtained, freeze‐dried, fixed in formaldehyde gas at 80°C for 1 h, embedded in vacuo in plastic (Durcopan Fluka) without immersion in any intermediary solvents, cut at 4 μm and covered with autoradiographic stripping film (Kodak AR 10). The auto‐radiograms were developed after 3 months. Rabbit retina was also incubated at 37°C for 15 min in a balanced salt solution (Ames, Parks & Nesbett 1980) with 2.5 μCi (3H)‐ADTN/ml. After washing for 2°5 min at 0°C the tissue pieces were processed for autoradiography as above.

Detection of radiolabelled bone marrow cells bearing surface ace immunoglobulins by combined autoradiography and immunoperoxidase

A method is described for the simultaneous detection of radiolabelled bone marrow cells bearing surface immunoglobulins by combined autoradiography and immunoperoxidase. Bone marrow cells from normal CBA mice prelabelled in vivo with 125IUDR or exposed in vitro to [ 3H]thymidine were incubated with rabbit anti-mouse immunoglobulins under capping conditions, washed, cytocentrifuged and treated with methanol and hydrogen peroxide to destroy endogenous peroxidase. Cells were then covered with peroxidase-conjugated goat anti-rabbit immunoglobulins, washed, treated with diaminobenzidine a and hydrogen peroxide and finally covered with autoradiographic stripping film and exposed for different times. Peroxidase-positive cells were typically capped and those radiolabelled had autoradiographic silver grains overlying the nucleus.

Pyronin Y for staining stripped autoradiographs prepared from plastic embedded sections of rat tissues containing plutonium.

Autoradiography prepared by the stripping film technique from 1 μm sections of semithin plastic embedded liver and bone tissues were poststained for examination with a 1% pyronin Y solution. The use of this dye avoids heating the tissue section and the overlying photographic emulsion. It also allows the staining of the tissue section without excessive uptake of the stain by the gelatin of the stripping film.

Methodological basis for an autoradiographic demonstration of insulin receptor sites on the surface of whole cells: a study using light- and scanning electron microscopy.

SUMMARYAn autoradiographic technique is described which allows, on whole cells, the determination of the number of receptor sites which are occupied by 125I‐insulin according to defined binding conditions. Glutaraldehyde fixation prevents the dissociation of the bound tracer from the receptor, which is the basis for a quantitative correlation between biochemical and autoradiographical data.The homogeneous coating of whole cells with a thin emulsion layer, which was required for these experiments, was achieved by a stripping film technique. An accurate judgment of the coating was possible only with scanning electron microscopic (SEM) techniques. As observed in the SEM, the photographic layer was smooth, uniform and in good contact with the top of the cell. The signal measured in this area was analysed. The majority of the grains also originated from the top side of the cell surface, despite the evidently three‐dimensional distribution of the label. As determined in test specimens, 80–85% of the grains originated from 3–4 keV electrons. Due to the short range of these electrons, the grains represent the distribution of the label on the part of the cell surface which is in close contact with the emulsion layer. 15–20% of the grains originated from β‐decays with higher energies and add partially to the background. According to these data a determination of the number of receptor sites per unit area of plasma membrane (receptor density) is possible in the surface area of the cell top.Both light microscopy (LM) and SEM were used to analyse autoradiographs. LM analysis is possible in principle but the analytical facilities of SEM are yet superior for the identification of silver grains. The registration of the grains is further facilitated in SEM because of the very large depth of focus.

A Simple Autoradiographic Technique for Studying Diffusion of Water into Seeds

Visual observations on rates and modes of water penetration into black bean seeds and into wheat and sorghum kernels during conditioning were accomplished by an autoradiographic procedure that eliminates a freezing microtome and liquid and stripping film emulsions. Seeds soaked in tritiated H(2)O were hand sectioned before freeze-quenching in liquid N(2) and subsequent block autoradiography on nuclear medicine film.

Convenient and rapid fluorescent staining of plant cell nuclei with ‘33258’ Hoechst

A simple and direct procedure is described which allows the fluorescent staining of plant cell nuclei and chromosomes with the fluorescent dye ‘33258 Hoechst’. This staining procedure is particularly suited for the subsequent determination of the mitotic index of plant cells grown as cell suspensions in liquid medium. It can also be used to stain nuclei after photographic processing of autoradiographs in the microautoradiographic detection of the incorporation of tritiated thymidine into nuclei with the stripping film method.

The Image Analysis System (CIAS) at the Canada Centre for Remote Sensing

SUMMARYAt the Canada Centre for Remote Sensing (CCRS) we have recently developed a new image analysis system (CIAS). This system is used both by CCRS scientists and outside investigators to analyze remotely sensed imagery from aircraft and satellite platforms. This paper describes the hardware and software architecture of CIAS, some algorithms for image correction, enhancement, and classification, and provides examples of output products from the system.The CIAS is composed of two sub-systems, the CIAS-70 sub-system configured around a modified General Electric IMAGE 100 system6 and the CIAS-40 subsystem centred around a PDS colour microdensitometer. In addition to major modifications to the IMAGE 100 hardware, we, in association with Computing Devices company and DIPIX, have also designed and implemented an array processor optimized for classification. This image analysis processor enables one to classify, for example, a full LANDSAT frame (7 × 106 pixels; four bands) into 93 classes with maximum likelihood discrimination in less than 14 minutes. Three separate data paths permit parallel image processing activity to occur. One of the large disk drives is dual-ported and interfaced to both the CIAS-70 sub-system's PDP-11/70 minicomputer and to the PDP-11/40 minicomputer of the CIAS-40 sub-system. The three-colour flatbed read/write microdensitometer can be used, for example, to quickly scan an aerial photograph for analysis on the CIAS-70 subsystem. A high speed interprocessor communication link ensures that tasks on both systems which require shared resources are correctly sequenced.Image input media are computer compatible tapes in CCRS-JSC format9, EROS Data Center formats, or photographs. A graphics tablet is used for limited digitization of map information, selection of map information, selection of training and test sites, and for map overlay. Output products are single-class plots scaled to approximately match map scales from 1:50,000 to 1:1,000,000, colour photographs produced on the PDS microdensitometer, the CCRS colour strip film recorder, or the CCRS Image Processing System, tapes in CCRS-JSC format, and tabular listings of statistical results, such as class acreages. Examples of some of these products are shown together with procedures for outside investigators to obtain access to this equipment for remote sensing image analysis.

Experimental analysis of the deformation of convex cylindrical surfaces

The described method is based on the moire technque. By applying a special stripping film with a copied grid to a convex cylindrical surface, it is possible to express the equations of the deformed as well as the undeformed grid. By using the double-exposure photographic technique, a field of moire fringes will be obtained, which gives information of the component perpendicular to the direction of observation of the deformation vector in the plane perpendicular to the axis of revolution. By observing two different directions α1 and α2, the experimental data yield the calculation of the displacement vector. The application of the derived method and its reliability will be demonstrated by several examples. The method does not require highly expensive laboratory equipment and is especially useful for engineers in consulting and structural design.

Identification of T and B lymphocytes in pigs by combined E-rosette test and surfage Ig labelling

A procedure for combining labelling of B lymphocytes with [ 125I]anti-Ig-antibodies and E-rosetting is described which permits simultaneous detection of B lymphocytes and T lymphocytes. Pig lymphocytes were incubated with iodinated anti-Ig antibodies and the E-rosette test with SRBC was performed. E-rosettes appeared stable after methanol fixation of cell smears on agarose-coated slides and treatment for autoradiography on stripping films. The percentage of cells classified as B or T lymphocytes in pig peripheral blood closely resembles that in man. No doubly marked cells were demonstrated provided cells were classified as E-rosettes only when 5 or more red cells were attached.

High-speed autoradiography of 3H-thymidine-labelled nuclei.

High-speed autoradiography with stripping film of 3H-thymidine-labelled cells was tested. The tests involved: (a) various times of immersion of emulsion-covered cell preparations in the mixture of dioxane-PPO-POPOP, at 20 degrees C, (b) exposure of cell preparations and blanks for various times at either -70 degrees C or +20 degrees C, with different humidity levels. Autoradiographs of good quality could be produced by 2-min immersion in the scintillator, exposure time greater than or equal to 1 h at either temperature and relative humidity 20--30%. A linear relationship between autoradiographic efficiency and exposure time of 1--7 h was found at either temperature, although the efficiency of autoradiographs exposed at -70 degrees C was by approximately 30% higher than that of autoradiographs exposed at +20 degrees C. Background values of autoradiographs dried with a fan and exposed for 1/4--7 h at either -70 degrees C or +20 degrees C were 0.6--0.8 grain/100 micron2. Theoretical calculations and experimental data showed that high-speed autoradiographs are 30--50 times more efficient as compared with conventional stripping film autoradiographs, thus allowing a shortening of the respective exposure time. Theoretical aspects of efficiency and resolution of high-speed autoradiography are considered.

Wound healing of the brain of rats after cryonecrosis. Autoradiographic investigations with 3H-thymidine.

Histologic and autoradiographic studies were performed to investigate the cellular reactions during wound healing of the brain of rats after cryonecrosis. A parietal lobe was frozen for 30 s with a cryoprobe at a temperature of -196 degrees C. The survival time ranged between 12 h and 21 days. One h before killing, tritiated thymidine was injected intraperitoneally. Stripping film autoradiograms of the brain sections were made in the usual manner. A typical cryonecrosis develops 12 h after local freezing surrounded by an edematous tissue layer with activated mesenchymal and neurologlial cells. After 10 days a pseudocyst develops surrounded by highly cellular glial tissue. The pseudocyst is still recognizable 3 weeks later but without any remarkable cellular reaction. Autoradiographically the labeling indices of the fibroblasts, leptomeningeal,and endothelial cells in the periphery of the necrosis and the labeling indices of neuroglial, perivascular connective tissue, and microglial cells in the perinecrotic zone increase after 12 h and have their maximum between the 2nd and 3rd postoperative day. In the nonfrozen contralateral cerebral hemispheres, the labeling indices of neuroglial and mesenchymal cells show small peaks in the first 3 postoperative days,also. This can be explained by accompanying edema. There are no remarkable differences between frozen and nonfrozen parts of the brain 3 weeks after local freezing. The results underline the rapid repair of cryonecrosis.