Polytene chromosomes are present in many larval tissues in Drosophila. They result from subsequent rounds of DNA replication not followed by cell division, and they represent a material of choice to determine by cytological methods whether a particular DNA sequence is associated with a protein of interest. They can be most easily prepared from salivary glands of third instar larvae, where the degree of polytenization is maximal.
We previously described a method that combined protein immunostaining with fluorescent in situ hybridization (FISH) in order to allow simultaneous visualization of a protein of interest and a specific DNA sequence (Dejardin and Cavalli, 2004; Lavrov et al., 2004). Compared with previous methods, where FISH and immunostaining were performed in separate experiments, this approach has the great advantage of directly showing the co-localization of a protein with the locus of interest. However, this combination raises a number of technical difficulties. In some cases, the antibody immunostaining does not survive the FISH procedure. In the published protocol, we provided a set of solutions for this problem. In the worst situation, where the immunostaining signal can not preserved from degradation during FISH, one has to perform immunostaining, acquire many chromosome images under conditions in which it is possible to keep the record of the XY position of the microscope stage, then perform FISH, find the same chromosomes by repositioning the slide in the same XY coordinates as for immunostaining, acquire the FISH images, and finally superimpose the immunostaining and the FISH images using software such as Adobe® Photoshop® (Lavrov et al., 2004). Although this procedure is straightforward, it is quite labor intensive.
Here, we present an adaptation of the protocol described earlier (Lavrov et al., 2004). The main difference is that we reversed the order between the main steps. The FISH protocol is thus applied before immunostaining. This procedure is the simplest solution to the problems due to loss of immunostaining during the subsequent FISH procedure. For this reason, we recommend beginning with this protocol when analyzing the binding of your favorite protein on a specific DNA sequence present in polytene chromosomes.
However, it is possible that this protocol may not work for all epitopes (we have not yet encountered this problem), since the formaldehyde fixation of the chromosomes, performed before FISH, might not be sufficient to maintain the protein of interest anchored to the chromosomes throughout FISH. In this case, it is advisable to go back to the reverse procedure (Lavrov et al., 2004; i.e. Immunostaining first, then FISH).
Below we describe a three-day protocol, with the DNA hybridization performed first (overnight between the first and the second day), and the incubation with the antibody against the protein of interest performed overnight between the second and the third days. The method consists of five parts:
- Preparation of third instar larvae for extraction of salivary glands,
- Chromosome squashes from salivary glands,
- 3. In situDNA hybridization to polytene chromosomes,
- Immunostaining with a specific primary antibody,
- Immunodetection of both FISH and immuno signals using specific secondary antibodies.
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