Direct Fluorescence In Situ Hybridization to Polytene Chromosomes

Fluorescence in situ hybridization (FISH) and its modification, polytene in situ hybridization (PISH) (1,2,4), have widespread application in the analysis of gene localization on interphase, metaphase and polytene chromosomes. However, available protocols use the indirect fluorescence approach, which requires the tedious and timeconsuming immunological detection step in which a fluorochrome-conjugated secondary antibody is applied to detect or enhance the signal of a nonradioactively labeled DNA probe. Recently, the new methods for FISH (with the detection sensitivity of 50– 100 kb on human metaphase chromosomes) were introduced, in which fluorescein-11-dUTP was used to produce by nick translation the fluoresceinated DNA probes (5). We describe the modification of PISH (1)—a simple and rapid protocol for direct FISH to polytene chromosomes—that eliminates the lengthy immunocytochemical amplification step, has detection sensitivity around 4 kb and allows the analysis of the results in one hour after finishing the hybridization step. The salivary glands of Drosophila larvae were dissected in 1% sodium citrate and squashed under the coverslip in the drop of 60% acetic acid or dissected and squashed in 45% acetic acid on uncoated or silane-coated microscope slides (Polysciences, Warrington, PA, USA). Slides were placed on dry ice for 20–30 min. After removal of the frozen coverslip, the slides were rinsed in 100% ethanol and air-dried. Either in situ hybridization was performed on the same day or samples were stored at 4°C or frozen at -20°C for up to three days without any visible decrease in the intensity of the hybridization signal. The following probes were used in our study: Sciara coprophila rDNA (a gift from S.A. Gerbi, Brown University) (3), Drosophila cCS-1, which is a 30-kb cosmid from the 17C (Bx-fu) region of the X chromosome and Drosophila pCASPER2 containing 4 kb of the white gene from the 3C region of the X chromosome (both were gifts from William Mattox, M.D.A.C.C., University of Texas). One microgram of DNA was denatured by heating for 10 min in boiling water and quickly chilling on ice. Denatured DNA was random-primed and labeled using the fluorescein labeling mixture from a PCR labeling kit (Boehringer Mannheim, Indianapolis, IN, USA) (2 μL of 10× PCR mixture were used for a 20μL reaction), a hexanucleotide mixture and a Klenow enzyme from a nonradioactive DNA labeling kit (Boehringer Mannheim) for 16 h at 37°C according to the manufacturer’s protocol. The quantitation by slot-blot analysis showed that the yield of the labeled