Detection of Radioactive DNA in Polyacrylamide Gels.

Bands of radioactive DNA separated by polyacrylamide gel electrophoresis may be detected by autoradiography or phosphorimaging. Analytical polyacrylamide gels containing radioactive DNA are usually fixed and dried before autoradiography. However, if bands of radioactive DNA are to be recovered from the gel, the gel should generally not be fixed or dried.

Measurement of cell adhesion and migration using phosphor-screen autoradiography.

This study demonstrates the use of phosphor-screen autoradiography as a means of measuring cell adhesion and cell expansion on polymer surfaces. The method has particular merit in cases where a specific substrate may be opaque or biochemically incompatible with colorimetric assay methodologies. With the phosphor-screen autoradiography method, there was a linear relationship between cell number and quantitated radioactivity. The technique has also been validated by comparison with a colorimetric assay of adhesion conducted for attachment to conventional culture substrata. The data supported the view that the use of phosphor-screen autoradiography was a valid method for detecting cell attachment, and it gave equivalent results to the colorimetric assay. Furthermore, a comparison between phosphor-screen autoradiography and a dye-staining method showed that this technique can be used as a means of quantifying cellular expansion over surfaces.

A novel hinge system and incubation chamber for emulsion-coated coverslip autoradiography.

We describe an emulsion-coated coverslip autoradiographic technique for large (50 x 50 mm) sections of monkey or human brain. The technique uses adhesive-backed, teflon-reinforced aluminum foil as a flexible hinge that allows the coverslip to swing away from the slide so that the emulsion and tissue can be processed independently. We also describe a plexiglas incubation chamber that allows two slides with coverslips folded away to be processed back-to-back in 5 ml of reagent solution. In general use, the chamber minimizes the volume of solution needed to cover large slide-mounted sections and is ideal for radioligand or immunohistochemical reactions that use expensive reagents. For autoradiography, the chamber greatly facilitates independent processing of the emulsion and tissue section. Together, the foil-hinge method and incubation chamber virtually eliminate the major technical pitfalls of previous emulsion-coated coverslip methods. Moreover, they facilitate combination of histochemical or immunocytochemical staining with autoradiographic localization of neurotransmitter receptors in the same tissue section. This is demonstrated by thioflavin S staining of neuritic plaques and tangles and autoradiographic localization of serotonin 1A receptors in sections of Alzheimer disease brain.

Quantitative film autoradiography for tritium: methodological considerations.

The details of quantitative film autoradiography for tritium using tritium plastic standards were examined with respect to 3 issues: tritium tissue equivalent (TE) calibration; correction of autoabsorption differences for gray and white matter; and the use of carbon-14 standard sources as a method for quantifying tritium tissue images. Both 3H-tissue and 3H-plastic sources produced linear log-log relationships of 3H-concentration (nCi/mg source weight) vs source optical density (OD) over a reproducible OD range (0.080-0.800). The curves for both 3H-tissue and 3H-plastic were parallel; uncalibrated 3H-plastic standards exhibited a 4-fold higher autoabsorption over 3H-tissue sources for OD values in the linear range. Using chloroform extraction of brains from rats treated with either [14C]deoxyglucose ([14C]DG) or [3H]deoxyglucose ([3H]DG), we found neither isotope loss nor redistribution after defatting (30% reduction of tissue dry weight). After chloroform extraction, the OD values from both gray and white matter structures containing carbon-14 were unaltered. Gray matter OD values increased by 28.7 +/- 5.6% (mean +/- S.D.) in structures containing tritium; white matter structures containing tritium exhibited a 115.9 +/- 29.3% increase in OD after chloroform extraction. The increase in OD after chloroform extraction was a fixed percent for any given tritium OD value from unextracted tissue when the value was within the linear range of 0.080-0.800 OD units. The magnitude of the higher white matter autoabsorption for tritium was confirmed using tritium impregnated cow brain pastes of variable gray/white mixtures. Chloroform extraction of tissue from [3H]DG treated rats was therefore a suitable procedure for direct correction of regionally heterogeneous autoabsorption of tritium. Finally, the rates of image generation for tritium and carbon-14 sources were compared. The rate of increase of OD with increasing exposure time was found to be equal for 3H-tissue and 3H-plastic images; sources of carbon-14 in plastic, however, exhibited more accelerated rates of image generation when compared to tritium sources (i.e. 3H- and 14C-images did not covary with exposure time). The effect of non-covariance on tritium TE calibrated standards was the overestimation of OD values for 14C-plastic standards with increasing times of exposure (comparison of 4 week images to 1 week images showed errors of 35-40%). Use of carbon-14 sources to quantify tritium-generated images therefore required recalibration of 14C-plastic for all exposure times of interest.

The calibration of 35S or 32P with 14C-labeled brain paste or 14C-plastic standards for quantitative autoradiography using LKB Ultrofilm or Amersham Hyperfilm.

The relationship between 14C-labeled brain paste or plastic standards and 35S- or 32P-labeled brain paste was characterized with quantitative autoradiography. Film was exposed to both sets of 14C-labeled standards and 35S- and 32P-labeled standards for 15-148 h and the film was analyzed with computer densitometry. Subsequently, the tissue pastes were scraped from the slides, dissolved in tissue solubilizer and radioactivity determined by scintillation spectroscopy. A linear relationship was observed between the plastic and brain paste 14C standards as well as between both sets of 14C-labeled standards and 35S-labeled brain paste standards. The 32P-labeled standards demonstrated a poor linear relationship with the 14C-labeled standards and greater variability in the autoradiographic estimates than was seen with 35S or 14C. Results were similar for both Ultrofilm and Hyperfilm with all the radioisotopes. These findings demonstrate a simple linear relationship between plastic and brain paste 14C-labeled standards and between either 14C-labeled standards and 35S-labeled brain paste for use in quantitative autoradiography.

Quantitative whole-body autoradiography: recommendations for the standardization of the method.

The objective of this article is to identify the best conditions for preparing, handling, and exposing radioactive sections by using the Fujix BAS 2000 system for quantitative radioluminography. Regarding the influence of thickness of sections, thicker sections may allow shorter exposure times due to the increased radioactivity, but on the other hand they take more time for the freeze-drying process, resolution will be reduced, and the recovery of radioactivity will be lower due to increased self-absorption particularly in organs like bones or teeth. The pretreatment of the sections should depend on the method of exposure. Powdering with talcum is the most recommendable method when vacuum packaging the imaging plates and sections. Spraying with Nobecutan is recommended when using the cartridge method. Particularly for low concentrations, the vacuum-contact method should be the method of choice. To avoid a flare effect, the geometrical arrangement of the sections on the imaging plate (IP) should always be vertical to the scanning direction of the laser. An exposure time longer than 10 days is not recommended and the time between the end of exposure and start of scanning should be as short as possible. In order to reduce the background signal, it is necessary to expose the IPs in a shielding box in a cold environment. No positive chemographic effects of sections were found.