Preparation of iodinated standards for quantitative radioautography in vitro using a tritium-sensitive film

In the present study we developed iodinated 125I-standards and tested the response of tritium-sensitive sheet film (3H-Ultrofilm) to varying concentrations of 125I prepared from brain "paste" mixed with mono-iodinated TiTx gamma toxin. Results have practical implications since they allow quantitative measurements of autoradiograms obtained with 125I-coordinats. An example is shown for 125I-neurotensin binding sites on rat brain sections.     

Morphometry and cytometry correlated to quantitative autoradiography

Studies performed in various cell systems and designed to establish correlations between morphometric and functional parameters of individual cells are reviewed. Functional parameters were evaluated by utilizing quantitative 14C-autoradiography and measuring DNA, RNA and protein synthesis. Morphologic parameters were derived from the scanning of Feulgen-stained nuclei and the calculation of features related to shape, optical density and texture. A series of correlations between parameters of these two groups of features was established. Results such as the possibility of allocating cells to the G1, S and G2 phases by textural and densitometric features alone, without making use of the total DNA content and nuclear size, point to the power of this approach. The data, however, are not yet comprehensive enough to allow the interpretation of morphologic parameters in terms of cellular function on a large scale. It is emphasized that the measurement of the RNA synthesis rate will further promote the functional understanding of structural details and may help in making this approach useful for diagnostic purposes.     

Flat bed scanner in the quantitative assay of 35SO4-incorporation by X-ray film autoradiography of intervertebral disc sections

A rapid quantitation of proteoglycan synthesis distribution in intervertebral disc and endplates is described. Tissue blocks of disc (C7-Th1) in the midsagittal plane from ten female beagles were incubated in the presence of ³⁵SO₄ and prepared as histological slides. For comparison, sulphate incorporation rates in the C5–C6 discs were assayed by liquid scintillation. Autoradiographic film exposed against the labelled sections was developed and digitized for image analysis using a 256 grey level flat bed table scanner connected to a microcomputer. The film density versus dpm (disintegrations per minute) calibration was performed using a set of ³⁵SO₄-labelled glycosaminoglycan standards applied on the same film. Since section thickness, dpm calibration of the film density and the specific activity of sulphate in the medium were known, the incorporations per tissue volume could be calculated. The average incorporation rates of the anterior and posterior annulus fibrosus, nucleus pulposus and vertebral endplates were 5.2±0.9, 5.2±0.8, 4.5±0.6 and 4.1±0.8 pmol/mm³ per h (±SE, n=10), respectively and closely corresponded to those obtained by liquid scintillation. This method offers a convenient and reproducible way to measure the rate of proteoglycan synthesis in large tissue sections but also in thin cartilaginous tissues such as the vertebral endplate.     

Methods of quantitative autoradiography using incident light microphotometry.

Several different approaches of automated grain counting of microautoradiographic grain densities have been reported in the literature. Application of grain counters to cell biology is limited, however, primarily due to shortage of methods allowing the interpretation of grain counts on a molecular basis. Two suitable methods of quantitative autoradiography at the cellular level are reviewed, developed for the isotopes 14C and 125 I. They permit evaluation of absolute radioactivity in autoradiographs and, thus, determination of synthesis processes such as deoxyribonucleic acid synthesis, and of antigen densities on cell surfaces. In this approach towards quantitative autoradiography, grain densities are compared photometrically over labeled cells and over a standard source on the same autoradiograph. Allowance has to be made for the specific geometric factors of the isotope used. This can be advantageously done with an integrating type of measurement using incident light bright-field. With this type of recording, there is an exponential dependence of the photometric values on the radioactive dose. As an example of application, results are presented of the deoxyribonucleic acid synthesis rate of human myelocytes in aplastic anemia and of the immunoglobulin G density on lymphocyte membranes in the normal state and in chronic lymphocytic leukemia.     

Autoradiographic distribution of [3H]neurotensin receptors in rat brain: visualization by tritium-sensitive film

[3H]Neurotensin ([3H]NT) binds specifically to a single class of binding sites on slides-mounted sections of rat brain 1Kp = 5.1 nM; Bmax = 16.2 fmol/mg tissue). Bound [3H]NT can be displaced by nonradioactive NT and a series of its fragments and analogues with relative potencies that correlate closely (r = 0.89; p less than 0.01) to their potencies in the rat stomach strip bioassay. These results suggest that NT receptors are similar in both systems. [3H]NT binding sites were visualized by using tritium-sensitive LKB film analysed by computerized densitometry. [3H]NT receptors are highly concentrated in the external layer of the olfactory bulb, in the rhinal sulcus, in certain nuclei of the amygdala, in the substantia nigra, zona compacta and in the ventral tegmental area. The high density of [3H]NT receptors in the last two areas suggest an interaction between NT and brain dopaminergic systems such as the nigrostriatal and the mesolimbic pathways.     

Negative-staining autoradiography: a new technique for ultracryotomy utilizing an interposed film

A new radiocytochemical technique is reported for ultrastructural localization of diffusible substances, using negatively stained ultra-cryostat sections. A sheet of film interposed between the cryostat section and the emulsion layer has rendered negative-staining autoradiography (NSA) practical. The rationale of NSA is that the film completely shields the section from all moisture-producing autoradiographic processes, so that phosphotungstic acid (PTA) can stain the section either before or after autoradiography (ARG), without the possibility of ultrastructural damage by alkaline solutions, interference between PTA and photoprocessing compounds, and superimposed images of a gelatin layer stained with PTA. As a model to demonstrate the newly developed procedure of NSA, rat brains were labeled with [125I]-triiodothyronine, fixed with tannic fixative, immersed in a cryoprotectant, frozen in liquefied propane, and cryostat sectioned. The resulting higher yield of radioactivity (85%) on the section was confirmed by a radiation counter. The retention rate was approximately 20% greater than that of conventional sections. Developed silver grains were found on synaptic vesicles and mitochondria in the polymorphic layer of the dentate gyrus. In this report we will also discuss the problems associated with cryostat sectioning of fresh tissues, the concept of ARG resolution, the distribution pattern of developed silver grains, and the possible applications of NSA.     

A new technique for staining sections and recovering celloidin film in high resolution autoradiography

Ultrathin sections are stained immediately after cutting by placing them in contact with staining solution and then placed on a slide covered by a celloidin film. This method largely avoids precipitates of heavy metals. The recovering of celloidin film is improved using a stainless steel basket. This technique is far more reliable than that involving use of a filter paper.     

Calibration of the emulsion layers in quantitative electron microscopic autoradiography]

The quantitative results in electron microscope autoradiography are affected by many factors. The principal problem of this method is a low mechanization of all the operations made. This paper gives a short review of technical improvements. The use of a calibration permits to except those factors which exert their influence on the sensitivity of the method. Tritium sources were used for the determination of the sensitivity of the method.     

An approach to the theory of quantitative and double label autoradiography

The distributions of grains and tracks per cell, as functions of the isotope content in the cell, are basic to all quantitative autoradiography. In this paper a formulation of these distributions in terms of the distribution of grains per disintegration is suggested. An empirical method for evaluating this distribution for various isotopes and various geometrical relationships between source and emulsion is described. The results are used to examine the usefulness of different techniques for double labelling autoradiography.     

Standardization strategy for quantitative PCR in human seminoma and normal testis

Housekeeping genes are commonly used as endogenous references in quantitative RT-PCR. Ideally these genes are constitutionally expressed by all cell types and do not vary under experimental conditions. Tissues of 9 normal testes and 22 classical pure seminoma were obtained for RNA-extraction. Real-time RT-PCR was used to examine the mRNA-expression of ubiquitin C, beta-actin, GAPDH, 18S ribosomal RNA (18S rRNA) and porphobilinogen-deaminase (PBGD). Additionally, 3 normal testicular tissues and 39 seminoma, including 1 normal testis and 17 seminoma of the RT-PCR group, were utilized for microarray analyses. Ubiquitin C (protein degradation) was down-regulated, GAPDH (carbohydrate metabolism), beta-actin (cytoskeleton), 18S rRNA (ribosome) and PBGD (porphyrin metabolism) were up-regulated in seminoma. A normalization of the target gene data with up-regulated housekeeping genes would equalize or underestimate up-regulated data and overestimate down-regulated data. We demonstrate that none of the investigated housekeeping genes is suitable for normalization of the target gene RT-PCR data, but may be essential for tumor metabolism in human seminoma. Further, we developed a standardization strategy, which is applicable to many experimental investigations.     

Radioimagers as an alternative to film autoradiography forin situ quantitative analysis of125I-ligand receptor binding and pharmacological studies

Summary Three radioimagers, the μ-imager, the β-imager and the phosphorimager, were tested as alternatives to quantitative autoradiography on film, for receptor imaging and pharmacologicalin situ quantitative analysis. Two iodinated ligands¹²⁵I-interleukin-1α and¹²⁵I-gonadotropin releasing hormone agonist, were used for receptor characterization in mouse brain and pituitary sections. Due to the high number of the agonist receptors in rat pituitary gland, this tissue was used to compare measurements obtained from digital autoradiograms with classical γ detector determination. This permits the evaluation of radioimager efficiency and absolute quantification. Radioimagers represent an improvement in terms of time of image acquisition. All the radioimagers are more sensitive than film for the detection of low levels of radioactivity. The spatial resolution provided by the μ-imager compares favourably with that obtained on film autoradiograms while digital autoradiograms from the phosphorimager and β-imager did not show precise definition under our experimental conditions. Superimposition of histological structures from the stained sections with radiolabelled areas in the autoradiograms remains, at this time, the unique advantage of film. In conclusion, radioimagers represent an alternative to autoradiography on film or emulsion forin situ quantitative studies on tissue sections. They combine precise imaging forin situ binding studies with easy and direct access to counts in cpm. The improvement in radioimaging technology has, therefore, broughtin situ analysis of iodinated ligand binding to the level of accuracy that is obtained with classical detectors of radioactivity.     

Antibody accumulation in small tissue samples: Assessment by quantitative autoradiography

Uptake of radiolabeled ¹²⁵I monoclonal antibodies in small metastases can only be characterized by autoradiographic techniques. To obtain quantitative data out of autoradiographic images, a transformation of the essentially two-dimensional signal into the Bq per unit volume information is needed. Part of the calibration problem could be solved by using tissue-equivalent standard preparations. However, when aiming at a quantification of radioactivity in small areas (⩽- 2 mm diameter), special criteria had to be expanded upon for the reconstruction of the area in the dose matrix and for the correct integration of the radioactivity content.     

A quantitative collagen film collagenase assay for large numbers of samples

A new statistical procedure is described which permits the direct, quantitative comparison of alternative physical models for a set of experimental results. Its reliability is demonstrated through a computer simulation study, and its usefulness is illustrated in the analysis of nonlinear van't Hoff and Arrhenius plots for selected cases of steroid-protein binding and enzyme catalysis.