Autoradiography intensifying screens

Sensitivity of X-ray films can be increased by use of an intensifying screen or by pre-flashing. The following protocol describes autoradiography using intensifying screen and solid scintillation, respectively. 

Autoradiography cassette with intensifying screen (Genetic Research Instrumentation Ltd)... 

Fig. 7.1. Three types of autoradiography can be distinguished (i) direct exposure (direct contact of specimen with film, usually separated just by a Saran wrap for wet specimens) (ii) fluorography in which fluors are excited by the radiation resulting in multiple photons per -particle (iii) phosphor-intensifying screens which can absorb radiation under the emission of photons (most stable in the cold). The proper choice of method depends on the penetration level of the radioisotope and the intensity of radiation.

Labelled probe hybridized to target sequences on the membrane may be detected by autoradiography, p radiation emitted from 32p labelled nucleotides incorporated in the probe will expose X-ray film. Intensifying screens are used for initial exposures to amplify the image, exposures without amplification offer improved resolution but require much longer exposures even when high specific activity probes are used. 

Prior to autoradiography, the gel must be dried onto 3MM paper under vacuum at 60°C for 2 h, after fixing for 30 min. The gel is then exposed to X-ray film with an intensifying screen at -70°C (Notes 18 and 19). 

Place the air-dried nitrocellulose sheet into a cassette for autoradiography (12-72 hr at -80°C) using X-ray film and an intensifying screen. 

When dry, mark the plates with S-labeled radioactive or fluorescent ink and expose the plates to X-ray film or to a phosphorimager plate. If autoradiography is to be used, presensitization of the X-ray film with a flash of orange light to an optical density of 0.2 and exposure with an intensifier screen at - 70°C will greatly increase the sensitivity of detection of P-labeled peptides. 

Figure 1. Partial autoradiogram representing differential display of mRNA from MD cell suspension cultures. Lanes 1 and 2 are from cells cultured in 2% (w/v) sucrose, lanes 3 and 4 from cells cultured in 6% sucrose, and lanes 5 and 6 from cells cultured in 14% sucrose. All bands were obtained using the specific anchored primer T 2AA in combination with the primer 5 -CAGGCCCTTC-3. a-p2p]dCTP was used for radiolabeling of DNA. Autoradiography was performed using KODAK X-OMAT AR film for 2 h at - 80°C using one intensifying screen.

Perform autoradiography (1-5 h at RT using 35 X 43 cm Biomax MS film, Kodak, Rochester, NY, cat. no. 143-5726) in film casettes fitted with intensifying screens. 

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