We present a method and protocol for fluorescent in situ hybridization (FISH) in zebrafish embryos to enable three-dimensional imaging of patterns of gene expression using confocal laser scanning... Show moreWe present a method and protocol for fluorescent in situ hybridization (FISH) in zebrafish embryos to enable three-dimensional imaging of patterns of gene expression using confocal laser scanning microscopy. We describe the development of our protocol and the processing workflow of the three-dimensional images from the confocal microscope. We refer to this protocol as zebraFISH. FISH is based on the use of tyramide signal amplification (TSA), which results in highly sensitive and very localized fluorescent staining. The zebraFISH protocol was extensively tested and here we present a panel of five probes for genes expressed in different tissues or single cells. FISH in combination with confocal laser scanning microscopy provides an excellent tool to generate three-dimensional images of patterns of gene expression. We propose that such three-dimensional images are suitable for building a repository of gene expression patterns, complementary to our previously published three-dimensional anatomical atlas of zebrafish development (bio-imaging.liacs.nl/). Our methodology for image processing of three-dimensional confocal images allows an analytical approach to the definition of gene expression domains based on the three-dimensional anatomical atlas Show less
Sar, A.M. van der; Stockhammer, O.W.; Laan, C. van der; Spaink, H.P.; Bitter, W.; Meijer, A.H. 2006
In many implementations of DNA computing, reliable detection of hybridization is of prime importance. We have applied several well-established DNA mutation scanning methods to this problem. Since... Show moreIn many implementations of DNA computing, reliable detection of hybridization is of prime importance. We have applied several well-established DNA mutation scanning methods to this problem. Since they have been developed for speed and accuracy, these technologies are very promising for DNA computing. We have benchmarked a heteroduplex migration assay and enzymatic detection of mismatches on a 4 variable instance of 3SAT, using a previously described blocking algorithm. The first method is promising, but yielded ambiguous results. On the other hand, we were able to distinguish all perfect from imperfect duplexes by means of a CEL I mismatch endonuclease assay. Show less
