Fluorescence in situ Hybridization (FISH) Protocols and Applications
The design simplicity and cost-effectiveness of the early Fluorescence in situ Hybridization (FISH) protocols, combined with the significant acceleration of discoveries in related technical areas like fluorescence microscopy, digital imaging, and nucleic
- PDF / 1,916,107 Bytes
- 12 Pages / 504 x 720 pts Page_size
- 10 Downloads / 202 Views
1. Introduction Fluorescence in situ Hybridization (FISH) on histological sections allows visualization of whole chromosome territories or their subregions, as well as single copy genes, in cell nuclei within their native tissues (Fig. 1). Provided that an appropriate protocol is used, FISH on tissue sections is actually simpler than FISH on 3D-preserved cells (3D-FISH) (1–4). The first two issues important for FISH on sections are tissue fixation and the type of sections to be used. Most of the popular histological fixatives, in particular, Bouin fluid and glutaraldehyde, impede hybridization of nucleic acids. The fixative optimal for FISH is 4% formaldehyde. Paraffin and vibratome sections, when used for FISH, cause a number of serious problems, an impaired probe penetration and poor nuclear morphology, in the Joanna M. Bridger and Emanuela V. Volpi (eds.), Fluorescence in situ Hybridization (FISH): Protocols and Applications, Methods in Molecular Biology, vol. 659, DOI 10.1007/978-1-60761-789-1_5, © Springer Science+Business Media, LLC 2010
71
72
Solovei
Fig. 1. Examples of FISH on cryosections. (a, b) Four-color FISH on alveolar cells from the mammary gland of lactating mouse: highly transcribed casein (Csn3, white ) and a-lactalbumin (Lalba, yellow ) genes, house-keeping ribosomal gene (Rps27, red ), and transcriptionally silent rod opsin gene (Rho, green ). BAC probes for the genes were labeled with FITCdUTP (Rsp27 ), Cy3-dUTP (Csn3 ), TexRed-dUTP (Lalba ), and Cy5-dUTP (Rho ). m, milk droplet in the lumen of the alveolus, (b) shows a single alveolar cell nucleus with all eight signals at the larger magnification. (c, d) Simultaneous visualization of genomic region (blue) and histone modifications (green) characteristic for transcriptionally active euchromatin (c) and heterochromatin (d) in ganglion cells of mouse retina. The genomic region was visualized by FISH with BAC DNA (RP2474F9) directly labeled with Cy3; histone modifications were visualized before FISH using rabbit-anti-H3K4me3 (c) and rabbit-anti-H4K20me3 (d) antibodies (Abcam) and secondary donkey-anti-rabbit antibodies conjugated to Alexa488 (Dianova). Note that hybridized genomic region (a gene-desert on MMU2; arrows) does not co-localize with euchromatin (c) but clearly co-localizes with heterochromatin (d). Nuclei were counterstained with DAPI [blue on (a, b) and red on (c, d)]. All images are maximum intensity projections of 1–4 µm long stacks acquired with Leica SP5 confocal microscope equipped with five lasers: Diode (laser line 405 nm), Ar (488 nm), DPSS (561), HeNe (594 nm), and HeNe (633 nm). Scale bar on A is 10 µm; scale bar on B is 5 µm and applicable to (c, d).
first instance. In contrast, cryosections provide both a satisfactory nuclear morphology and a very good probe penetration (5). Furthermore, the use of thick (20–40 µm) cryosections assures that a reasonably high proportion of nuclei in the middle of the section remain intact, which is an important issue, for example, neurons with large nuclei. On the other hand, usin
Data Loading...
