Radioactive In Situ Hybridization to Animal Chromosomes

Although largely replaced by the use of fluorescent in situ hybridization (FISH) in animal and human molecular cytogenetics, the technique of radioactive in situ hybridization (RISH) still has some uses. Using practicable exposure times for autoradiographs of 3–4 wk, RISH is approx 50 times more sensitive than FISH using biotin- or digoxygenin-labeled probes, and probably 10 times more sensitive than the recently introduced system of tyramide FISH (NEN Life Sciences Prods., Code NEL 730/730A). In addition, the sensitivity of RISH can be increased with longer exposures, in a roughly linear fashion until the silver bromide grains in the emulsion approach saturation over the target; by contrast, FISH requires instantaneous expression of the signal. Because of its high sensitivity, RISH can be used with short probes (1 ), down to 200 bp, poorly labeled probes (2 ), short target sequences, and old slides (Table 1 ). Bands on the chromosomes can be excellent with RISH (3 ), requiring no enhancement by image analyzing systems, and observed with simple brightfield microscopy (Fig. 1 ); although finding a suitable batch of Giemsa stain can be difficult (see Subheading 3.6. and Note 1 ).