Tavi's Multicolor FISH Page
(fluorescence in situ hybridization)

Designed by OCTAVIAN HENEGARIU, Yale University
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MAIN TOPICS

FISH guide and troubleshooting: is a summary of the author's practical experience with numerous FISH procedures used or tested in time. This section contains over 150 images and close to 130 manuscript pages, which detail numerous factors that influence hybridization results (signal, background). There are tips on slide preparation, probe preparation, hybridization buffers, probe mapping, multicolor FISH, CGH, M-FISH, fiber FISH, RNA in situ, immunostaining and many other. The most important observations detailed here were published within several of the author's previous manuscripts.

Custom fluorescent nucleotide synthesis (Ref: Nature Biotech 18: 345-348 (2000) Get article here) describes an easy procedure for preparing fluorescent- or haptene-labeled dUTP and UTP. The simple chemical coupling reaction can be performed by anyone, and does not require background or training in chemistry or related areas. The custom-labeled nucleotides can be used in any procedure involving fluorescent DNA or RNA analysis (DNA sequencing, flow sorting, FISH, microarrays, etc), while reducing the costs of such procedures by 100-200 times. (Topics: 1. Chemical coupling of dye/dUTP; - 2. DNA labeling and purification; - 3. M-FISH labeling schemes;)

Improved cytogenetic slide preparation (Ref: Cytometry 43: 101-109 (2001) Get article here), describes a logical approach to improving chromosome spreading. It details a procedure, called "chemical aging", which helps achieve better hybridizations and shows that gradual denaturing of the slides helps preserve better chromosome architecture and banding pattern after FISH. (Topics- 1. Chromosome spreading; - 2. Chemical aging; - 3. Gradual denaturing)

Color changing karyotyping (CCK) (Ref: Nature Genet 23 (3):263-4 (1999) Get article here ) details a procedure allowing color karyotyping of human chromosomes without investments in new hardware or software. Any fluorescence microscope equipped with 3 filters is sufficient. Any camera currently available on your system can be used for image aquisition (digital photographic camera, CCD camera, cooled CCD camera). Image processing is done using a widely available image processing software (Photoshop, Adobe Corporation) on any computer. (Topics: 1. CCK principle; - 2. CCK examples; - 3. CCK methods/protocols)

Centromeric multiplex FISH (CM-FISH) (Ref: Lab Invest. 2001 Apr;81(4):475-81. Get article here) : describes a 2-hour FISH procedure, which uses combinatorial labeling of 6 fluorophores (similar to M-FISH), to identify aneuploidies and marker chromosomes on one slide, either on metaphases or on interphase nuclei.

Telomeric multiplex FISH (TM-FISH) (Ref: Lab Invest. 2001 Apr;81(4):483-91. Get article here): describes three FISH procedures, using combinatorial labeling of 3-5 different fluorophores (similar to M-FISH), to simultaneously detect all 41 human subtelomeric probes on the same slide. A similar approach for the mouse chromosomes (mTM-FISH) is also detailed.

Identification of all mouse chromosomes using BACs (Ref: Mamm Genome 2001, Vol 12, p462-465. Get article here). A triple color FISH procedure used to detect (on the same metaphase) all mouse chromosomes, using 40 mouse-specific BAC clones. Useful for probe/gene mapping. As there are only three fluorescence colors, the procedure can can be performed using any fluorescence microscope equipped with three filters.

DNA size measurements (Ref: Biotechniques 2001, Vol 31, p246-250. Get article here). A simple and rapid procedure for stretching DNA molecules on slides (using DNA cloned in vectors, such as plasmids, cosmids, BACs, PACs, YACs or from genomic DNA), and assessing the size of the various DNA fragments after YOYO staining.

Common fluorophores and dyes (Periodically updated)
Comprehensive fluorophore list (includes absorption and emission peak values - from Zeiss)

µArrays (DNA Microarrays analysis); guide and troubleshooting; complete protocols for hybridization, analysis.
Coming up !!

As many pages include images, if you are connected to the Internet by modem, please be patient while the page(s) load onto your computer. Please note that links will be activated only after data is published.

"Happy New Millenium", with "The Dancing Chromosomes"
Please note that all images are copyrighted.

Send me an e-mail at: Name:"octavian.henegariu", Server:"yale.edu" (please TYPE them in your email application using @ in between) or at Name:"ohenegar", Server:"yahoo.com" (please TYPE them in your email application)

Back to Tavi's Home Page; Last modified : June 05, 2001

Tavi's Multicolor FISH Page (fluorescence in situ hybridization)

Tavi's Multicolor FISH Page
(fluorescence in situ hybridization)