Absolute quantitative autoradiography of low concentrations of [125I]-labeled proteins in arterial tissue.

Abstract

We developed a method for absolute quantitative autoradiographic measurement of very low concentrations of [125I]-labeled proteins in arterial tissue using Kodak NTB-2 nuclear emulsion. A precise linear relationship between measured silver grain density and isotope concentration was obtained with uniformly labeled standard sources composed of epoxy-embedded gelatin containing glutaraldehyde-fixed [125I]-albumin. For up to 308-day exposures of 1 micron-thick tissue sections, background grain densities ranged from about two to eight grains/1000 micron 2, and the technique was sensitive to as little as about one grain/1000 micron 2 above background, which correspond to a radioactivity concentration of about 2 x 10(4) cpm/ml. A detailed statistical analysis of variability was performed and the sum of all sources of variation quantified. The half distance for spatial resolution was 1.7 micron. Both visual and automated techniques were employed for quantitative grain density analysis. The method was illustrated by measurement of in vivo transmural [125I]-low-density lipoprotein [( 125I]-LDL) concentration profiles in de-endothelialized rabbit thoracic aortic wall.

Characterization of sites with elevated LDL permeability at intercostal, celiac, and iliac branches of the normal rabbit aorta.

“…Uniformly labeled standards of 125 I-BSA were prepared according to the procedures of Schnitzer et al 29 Hautchen preparations and calibration slides were dipped in nuclear emulsion (Kodak NTB-2, Eastman Kodak Co, Inc) at 40°C and dried vertically on filter paper at room temperature and 80% relative humidity for 1 hour. 1429 Controls consisted of clean blank slides and slides with a Hautchen preparation of nonradioactive tissue.…”

Macromolecular transport within heart valves.

“…12 Standard calibration samples were dipped, exposed, and developed at the same time as each group of radioactive tissue slides. Nonradioactive tissue sections of rabbit aortic walls were included with each group on a separate slide as a control for positive or negative chemography.…”

“…Transvalvular concentration profiles for the l25 I-labeled proteins were determined by absolute quantitative autoradiography 12 and were compared with theoretical mathematical models based on fundamental transport principles. The best estimate for the relevant transport parameters was achieved using nonlinear optimization techniques.…”

Calibration of Densitometry in Radio-Isotopic in Situ Hybridization

“…Nowadays, the use of high resolution CCD cameras for image acquisition, has rendered one of the major objections against cytometry, the occurrence of a distributional error, obsolete: this error can either be considered to be irrelevant because the object size on the pixel of the camera is smaller then the optical resolution of the image acquisition system (Checo et al, 1994) and/or this error does not occur at all because silver grains in autoradiograms are fully opaque and, therefore, the transmittance is always equal to the fraction of coverage by grains (Jonker et al, 1997;Moorman et al, 2000). The use of bright field optical density (OD) measurements as a replacement for laborious grain counting has been validated for low radioactivity levels (Schnitzer et al, 1987). OD measurements were also shown to correlate well with tissue concentration of substances at higher radioactivity levels (Jonker et al, 1997).…”

“…The use of plastic calibration standards (Ito et al, 1995) has the disadvantage that the radioisotopes as well as the quenching conditions differ between standards and biological material. The same holds for dot blot standards on membranes (Palfi et al, 1998;Vizi and Gulya, 2000), which were introduced to avoid the efforts involved in preparing cryo-sectioned tissue paste standards (Miller, 1988) or embedded and sectioned gelatin standards (Schnitzer et al, 1987). Calibration spots on glass slides can be treated in parallel with the tissue sections and thus a calibration curve with the same radio-isotope can be obtained with the same quenching, tissue processing, and image acquisition conditions as the tissue sections (Larsson and Hougaard, 1994;Jonker et al, 1997).…”