FISH

Fluorescent in situ hybridization (FISH) is a molecular cytogenetic technique used to detect the presence of a specific DNA sequence on a chromosome.

The technique is based on the use of a probe: a short DNA sequence (10-25 nucleotides), specifically designed for the target sequence. To allow the detection of a wanted genic locus the probe is fluorescent-labelled (direct labelling) or can be rendered fluorescent in a subsequent phase (indirect labelling).

The first protocol step is the attachment of either interphasic or metaphasic chromosomes on a solid surface (glass); then the sample and probe solution is denatured and incubation starts. During the incubation period (hours, depending on the protocol), target DNA and probe interact and hybridize. To avoid background, unbound/partially bound probes are washed away, by repeated washes, after incubation.

The detection step can be different: in case of direct detection the probe is already fluo-marked and the sample can be directly observed at fluorescence microscope. On the contrary, in the indirect labelling it is necessary to add an enzymatic or immunological detection system, which will render the probe fluorescent. This method needs more time but allows to amplify the signal and get a better detection.

FISH is a versatile technique: can be useful to detect a gene locus on a chromosome, chromosomal abnormalities and rearrangements. Nowadays several connected techniques have been developed starting form the “classical” FISH, which is focused on chromosomal loci.

 (Giada Cipollina)

Immuno-FISH

As the name suggests, this is a combination of two techniques, the standard FISH, either on flattened chromosome preparations (2-D FISH) or on three-dimensionally preserved nuclei (3-D FISH), and the other indirect or direct immunofluorescence. Immunofluorescence permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc), so that both DNA and proteins can be analyzed on the same sample. Numerous methods involving a variety of fixation and permeabilization techniques can be used for immunofluorescence applications, and the choice depends on cell type, epitope, and antibody being used.

Some protocols establish that FISH procedure comes first, but not all antigens will be preserved after the various steps in the protocols, so it is possible to apply the primary or primary and secondary antibodies and proceed with a fixation step prior to the FISH procedure: in this way, the detected epitope will be preserved.

The combination of these techniques  is often used to investigate co-localization of genomic regions with proteinaceous entities within interphase nuclei such as nucleoli or promyelocytic leukemia (PML) bodies. It has helped to position chromosomes in interphase nuclei.

(Fabiola Varese)

RNA-FISH (Luca Torello, work in progress)

others....