Autoradiographic Preparations Research Articles

Light-dark-field double exposure photomicrographic method for autoradiographic preparations

Light-dark-field double exposure photomicrographic method for autoradiographic preparations

The applicability of grain density autoradiography to the quantitative determination of algal species production: A critique

Grain density autoradiography can be utilized to quantitatively determine the productivity of individual species in mixed phytoplankton assemblages but only after grain counts have been corrected for the effects of source geometry on efficiency as well as for the effects of spurious grain formation and erasure. The common simplifying assumption made by all workers to date, that the grain count over a cell is directly proportional to the cell’s radioactivity, is never realized with mixed species assemblages and such attempts at quantification are invalid. The procedure of apportioning total filterable radioactivity to the cells counted in autoradiographic preparations is also suspect. A method of data correction is presented which makes quantification feasible.

Cerebello-olivary fibers: their origin, course and distribution in the North American opossum.

Although degeneration techniques suggest that cerebello-olivary fibers are limited in their origin and distribution, horseradish peroxidase and autoradiographic experiments make it clear that they arise within all cerebellar nuclei and project to most, if not all, areas of the contralateral inferior olive. Autoradiographic preparations show that cerebello-olivary fibers are highly ordered and suggest that the dentate nucleus projects primarily to the principal olive, the interpositus anterior relays particularly heavy to the dorsal accessory nucleus and the interpositus posterior distributes extensively to the medial accessory complex. Evidence for a small projection from the fastigial nucleus to the caudal medial accessory nucleus is also available. However, it appears clear that neither the dentate nor the interpositus nuclei project to just one subdivision of the olive. For example, although dentate fibers end extensively within the principal nucleus some of them also distribute to portions of the medial accessory nucleus and perhaps the dorsal accessory nucleus as well. The medial accessory olive is particularly complex and at rostral levels receives input from both interposed and dentate nuclei, whereas more caudally it receives a projection from the fastigial nucleus. Olivary fibers from both the interposed and dentate nuclei traverse the brachium conjunctivum descendens and distribute primarily to the rostral 2/3 to 3/4 of the olive, whereas those from fastigial neurons take a different route and end more caudally. Experiments utilizing horseradish peroxidase as a retrograde tracer suggest that cerebello-olivary fibers from both the interpositus anterior and dentate nuclei take origin from a population of generally small neurons.

Autoradiographic demonstration of human basophils

A new technique is described which enables a selective tinctorial visualization of human blood basophils in autoradiographic preparations. Consecutive administration of 5-aminoacridine hydrochloride and aldehyde fuchsin rendered a sufficient preservation of basophils preventing leaching of their water soluble granules. Extensive control tests provided sufficient evidence to exclude chemographic artifacts produced by other histochemical precedures. The technique eliminates the obstacles to study the kinetics of human blood basophils and their realtionshp to atopic hypersensitivity reactions applying tritiated thymidine and radioiodine labeled IgE or corresponding antisera.

Transcription sites in spread meiotic prophase chromosomes from mouse spermatocytes.

Mouse spermatocytes at pachytene stage have been examined by whole-mount electron microscope techniques complemented with autoradiography as an approach for visualizing their transcriptive activity. Structural elements of meiotic bivalents, such as synaptonemal complexes and chromatin fibers, have been satisfactorily displayed in the total set of autosomal and sexual bivalents in single spermatocytes. Adequate preservation of the entire set of bivalents has provided a basis for recognition of sites where presumptive preribosomal RNA and heterogeneous nuclear RNA species are being transcribed at different segments of autosomal bivalents. Nucleoli attached to the basal knob region where nucleolar organizer cistrons are assumed to be located and ribonucleoprotein fibrils associated with distinct chromatin loops have been recognized. These structural findings have been correlated with display of [(3)H]uridine incorporation sites in thin-section and whole-mount electron microscopy autoradiographic preparations. A low transcriptive activity of the sexual bivalent contrasted with extensive gene expression in autosomal bivalents. Each sex chromosome shows a double axial core. A short region of pairing with a synaptonemal complex joins the two chromosomes at one end. We conclude that variations in the rate of RNA synthesis throughout meiotic prophase stages in the mouse are expressed as fluctuations in the amount and distribution of distinct RNA species at specific segments of the bivalents.

Placental transfer of streptozotocin in the rhesus monkey.

The induction of a diabetic-like state by the drug streptozotocin (Stz) has been useful for studies on the effects of maternal glucose intolerance upon the monkey fetus. However, the question of transplacental passage of Stz and its effect upon the fetus has to be considered if the drug is to be administered post-conception. Experiments in which Stz was administered maternally and then analyzed in maternal and fetal blood by disc-plate assay indicated prompt movement to the fetal circulation. Slow maternal degradation of Stz was associated with higher fetal blood levels of the compound. Studies with 14C-streptozotocin suggest that about one half of the radioactivity present in maternal and fetal plasma following intravenous maternal administration is intact 14C-Stz; after one hour, only one fifth of the radioactivity is still associated with the Stz molecule. By the time maximum fetal levels of the drug were achieved, maternal levels of Stz had decreased to one sixth and one tenth of initial levels. Fetal levels did not exceed one half to one third of the simultaneous maternal plasma concentration, implying placental limitation to transfer of streptozotocin from mother to fetus. No specific localization of 14C-Stz by pancreatic beta cells was observed in either paraffin or frozen-dried autoradiographic preparations. No evidence of high levels of uptake of 14C-Stz were found in pancreatic tissue in comparison to other selected fetal tissues. Although Stz does cross the hemochorial placenta to a limited extent, it appears to have no discernible effect upon the fetal pancreas.

Autoradiographic localization of 3H-GABA in the cockroach brain

1. 1. Tritiated gamma-aminobutyric acid is taken up from the medium by in vitro incubated brain slices of Periplaneta americana. After 45 min incubation more than 96 per cent of the radioactivity contained in deproteinized tissue extracts was accounted for by unchanged 3H-GABA. 2. 2. Localization of the uptake was studied autoradiographically. Dense accumulations of reacted emulsion grains were observed over well-defined nerve cell groups (e.g. in the pars intercerebralis anterior), nerve fibre bundles (e.g. a tract merging in the central body) and neuropilar structures (e.g. lower part of the central body, antennal glomeruli). 3. 3. No appreciable differences were observed in autoradiographic preparations from tissues incubated for 15, 30 and 45 min.

An autoradiographic and immunofluorescent study of the infectious cycle of sheeppox virus in cell cultures exposed to 5-fluorodeoxyuridine.

No progeny virus is detected by determination of the infectivity titres when embryo muscle cell cultures are exposed to a concentration of 10−5 M of 5-fluorodeoxyuridine at the time of sheeppox virus infection. The autoradiographic preparations of FUdR treated cells showed cytoplasmic labelling into viral DNA (tritiated deoxyuridine) in 4 to 9% of the cells during the first 24 hours of infection. In cells exposed to FUdR in which viral DNA synthesis was detected by autoradiography, progeny virus not detectable by infectivity test might be produced. The demonstration of viral proteins by immunofluorescence served to establish that viral DNA synthesis is not essential for viral protein synthesis in FUdR treated cells. Under light microscopy, there is no apparent difference between the cytopathic effects of viral infection in treated and in untreated cells.

A modified technique for producing “En face” (Häutchen) preparations of endothelium for autoradiography

Because endothelium is a layer only one cell in thickness, any normal cross- or longitudinal-section of a vessel wall can only include a few endothelial cells the nuclei of which may not be visible in the preparation. The search for isotopically labelled cells in such sections is, therefore, extremely slow and tedious. The production of suitable sheets of endothelium to be looked at en face reduces the work and makes isotope recognition and grain counting considerably easier. Since en face preparations were first used by ZAHN 1, the most successful techniques have required the embedding of the endothelium in a base such as nitrocellulose and the tearing away of the other layers of the vessel from it. Such preparations are termed h~utchen or little skins and are one cell thick. POOLE et al. 2 briefly reviewed other methods and gave details of their nitrocellulose embedding technique. Subsequently POOLE3, a published an excellent autoradiograph of a h~tutchen preparation, but he gave no indication of the method used. For this last reason we wish to set out our technique in this preliminary note before publishing quantitative experimental findings. The production of h~tutchen by the nitrocellulose embedding method is simple, but we have found it unsatisfactory for autoradiography. The cellulose base lifts from the slide and crinkles during drying of the autoradiographic film, thus introducing distortion and failure of register between the cell preparation and the photographic emulsion. A modification using formvar in place of nitrocellulose was equally unsatisfactory for though there was no crinkling, moisture was trapped beneath the formvar, causing a high background in autoradiographic preparations. Frozen tissue stripping 5 has also been tried with unfixed material but the areas of endothelium obtained were too frequently small and fragmentary and hence unsuitable for autoradiography.

Cell proliferation in the regenerating liver of continuously irradiated mice.

The effect of prior continuous irradiation at dose-rates of from 16 to 70 rads per day for up to 100 days on the regenerative capacity of the liver was examined in young C57BL mice after partial hepatectomy in order to investigate the extent to which a slowly proliferating cell renewal system accumulates, stores and repairs lethal and non-lethal latent cellular radiation injury affecting the pattern of cell proliferation and thus the speed and efficiency of regeneration. Radiation effects on DNA synthesis and cell division were determined by analysis of incorporation of labelled DNA precursor (tritiated thymidine) and changes in mitotic indices in autoradiographic preparations. Comparison of changes in liver weight, labelling and mitotic indices and nuclear grain counts revealed that prior continuous exposure delayed the appearance of peak activity of proliferation of the parenchymal and littoral cell populations for periods up to 24 hours. This inhibition was dependent on dose-rate, duration of exposure and accumulated dose. At the higher dose-rates and after long periods of irradiation there were (1) a decrease in the rate of entry of cells into synthesis and mitosis, (2) a decrease in the number of hepatocytes taking part in the regenerative process, (3) an increase in the overall duration of cell proliferation during regeneration, and (4) increase in the number of observable chromosome aberrations and possibly of polyploid parenchymal cells. In spite of relatively large accumulated radiation doses, cell proliferation was initiated and maintained to restore most of the liver mass during the period of observation, particularly at the lower dose-rates.

An autoradiographic study of incorporation of labelled 5-hydroxytryptophan and 5-hydroxytryptamine in the brain

In order to investigate the histochemical localization of serotonin in the central nervous system, light and electron microscopic autoradiography has been studied by using labelled 5-hydroxytryptophan (5-HTP) and 5-hydroxytryptamine (5HT).Light microscopic autoradiography was carried out as follows: C14-5-HTP (0.5μc/g) was injected intraperitoneally to the mice pretreated with Catron (0.01mg/g) 5 hours before the administration of C14-5-HTP. C14-5-HT (0.5μc/g) was injected intraperitoneally to the another group of mice without pretreatment. The animals were sacrificed at various time intervals up to 24 hours after injection of labels and autoradiographic preparations were made with kodak NTB2 by dipping method.For electron microscopic autoradiography, H3-5-HTP (5μc/g) was injected intraperitoneally to the rats. One hour after the administration of labelled 5-HT, the rat brain was fixed by OsO4 immersion after glutaraldehyde perfusion and ultrathinsections were coated with Sakura NR-MI by wire-loop method.An autoradiograph of brain after intraperitoneal injection of C14-5-HT revealed that 5-HT was taken up by the paraventricular structures and the neighbouring small area, but not by the ordinary brain regions. An autoradiographic localization of labels following intraperitoneal injection of 4-5-HTP was revealed markedly in autonomic area and limbic system, in addition to paraventricular structures. In as short a time as 5 minutes, marked uptake of C14-5-HTP was observed in the neuropil, while less uptake in the cytoplasm and nuclei. Because the majority of labels in the neuropil is supposed to be due to 5-HT, 5-HT may probably be synthesized and matabolized in the synaptic nerve endings, probably not in the cytoplasm of the cell bodies. Electron microscopic autoradiograph of the nucleus caudatus and nucleus ventromedialis hypothalami one hour after intraperitoneal administration of H3-5-HTP showed that two-thirds of the total silver grains were located in the neuropil and a third in the cell bodies. In the neuropil a half of the grains was localized in the presynaptic nerve endings, having a tendency to be concentrated in the presynaptic terminals containing granulated vesicles.

Methods for the preparation of autoradiographs with coverslip cultures.

AUTORADIOGRAPHIC procedures are being increasingly used in all branches of cell biology. The routine methods are extremely useful with sections and air dried cell suspensions1–4, but the extension of these methods to coverslip cultures has often been unsatisfactory. The usual procedure for coverslip preparations involves cementing the coverslip cell side up on a slide in a drop of mounting medium (usually ‘Permount’) before covering with liquid emulsion or AR 10 stripping film5. This method often causes opaque emulsion patches in the space between coverslip and slide and the coverslip quite frequently floats off during rehydration. This communication describes a method which eliminates some of the problems encountered in autoradiography on coverslip cultures.

Cell population kinetics in the epithelium of the forestomach of the mouse

The dynamics of cell renewal in the slowly proliferating epithelium of the mouse forestomach were analysed by the Method of Labeled Mitoses (pulse labeling with tritiated thymidine, serial killing, preparation of autoradiographs, determinations of fraction mitoses labeled between injection and sacrifice). The following values were estimated: T M about 1–2 hr; T G2 about 1–2 hr; T S about 13.5 hr. The estimate of T S is higher than that reported for more rapidly proliferating tissues and indicates that T S is not a true constant in all mouse tissues. Nevertheless, it was suggested that differences in cell cycle times may reflect primarily, but not exclusively, differences in T G1 and T G0 . Proliferative cells of the forestomach may undergo one of two possible proliferative cycles. Post-mitotic cells may either pass directly into a G 1-phase ( T c of about 30 hr) or be shunted into a moderately long G 0 -phase ( T C of about 2 to 4 weeks). The significance of this finding to the concept of cycle time is briefly discussed. Examples of G 0 cells in a few proliferating systems are cited.

On the replicative organization of DNA in the polytene chromosome of Drosophila melanogaster

The appearance of discrete labeling sites on autoradiographic preparations of pulse-labeled Drosophila salivary gland chromosomes indicates the presence of several independent points of DNA synthesis along the length of these polytene structures. While subject to an alternative interpretation, the data are most simply reconciled with the presence of several molecular ends in the DNA complement along the axis of each chromosome arm.

ELECTRON MICROSCOPIC AUTORADIOGRAPHY

An improved method has been developed for the preparation of autoradiographs for electron optical study. The refinement lies principally in the routine production of uniformly thin layers of photographic emulsion over the tracer-labeled cell sections. This is accomplished by means of a centrifugal spreading mechanism. Maize root tips grown in the presence of H(3)-thymidine were examined electron microscopically by this technique and were found to display nuclear labeling with impressive clarity. The new procedure utilized here yields objects in which the finest cell structures are easily resolvable without recourse to gelatin digestion techniques.

Proliferative cycle in the growing hair follicle of the mouse.

THE intermitotic phase has been divided into three segments: the phase of synthesis of deoxyribonucleic acid (DNA) (S phase), preceded and followed by ‘gaps’ called G 1 and G 2 respectively1. The duration of these phases can be accurately measured by pulse-labelling with tritiated thymidine, followed by serial killing, preparation of autoradiographs, and determination of the fraction of mitoses labelled at various intervals between injection and death2,3. Several kinds of cells have been so studied; the present communication adds to the list the cells of the growing-hair follicle in the mouse.

Radioactivity experiments for high schools using orange glazed ceramics

Describes the preparation of autoradiographs and the measurement of radiation generated by glazed ceramic dishes.

Controlled staining of autoradiographs.

The preparation of autoradiographs in which the tissue and the emulsion are in permanent register is often complicated by staining after the photographic image has been developed and fixed. While general oversight methods can be satisfactory, controlled, specific staining can be obtained with most basic dyes when the pH is properly regulated. The reactivity of the gelatin is suppressed at a pH of 4 or slightly below whereas nuclei, ergastoplasm, cartilage, mast cells, mucus, etc. stain readily. Basic fuchsin,. 05% at pH 3.5-4.0 in dilute (1:10) McLlvaine buffer, is recommended. The final preparation contrasts in color and transparency with the black, opaque silver grains.

The stripping-film technique of autoradiography

Stripping film either with a permeable base or an impermeable base is used for the preparation of autoradiographs (ARG). The procedure for the latter kind of stripping film has been adequately described by Boyd and Williams, (1) and this communication deals entirely with permeable-base stripping film as used in this laboratory. Many details of the technique have been developed empirically, and in many instances the reasons for adopting a certain procedure or discarding another have not been adequately checked. The type of stripping film which is now in use was designed in collaboration with the Research Laboratories of Messrs. Kodak Ltd. (2, 3) The technique itself has not been changed since its introduction, but much detailed work has been done to avoid artifacts and ensure consistent results in applications to biological problems.

Limitation in the use of tissue slices for the study of metabolic phenomena

The use of tissues slices for metabolic studies gives results valuable only from the qualitative view-point. The histochemistry of liver slices reveals indeed that there is a disappearance of the basophilia of the inner layers of cells and, consequently, autoradiographic preparations of slices incubated with tagged glycine show that the incorporation of the amino-acid is limited to the outer layers of cells.