Choosing the Right Labeling Method for your Hybridization Experiment Choosing the right labeling method for your hybridization experiment. Includes: Homogeneous labeling methods for DNA; Homogeneous labeling methods for RNA; Stability of probe-target interaction; Nonradioactive labeling of oligonucleotides; Double-stranded versus single-stranded probes.
Combined DNA In Situ Hybridization and Immunocytochemistry Protocol Protocol for combined DNA in situ hybridization and immunocytochemistry for the simultaneous detection of nucleic acid sequences, proteins, and incorporated BrdU in cell preparations. Includes: Cell preparations and BrdU labeling; Detection of antigen by immunocytochemistry (ICC); Visualization of ICC antigen; -Gal-BCIG reaction (for producing a blue precipitate visible under brightfield microscopy); Cell processing for in situ hybridization; In situ hybridization (ISH); etc...
In situ Hybridization Describes the basic principles of in situ hybridization and advantages and disadvantages of different methodologies that can be used. Includes: Probe Selection; Probe Generation; Probe Labels; Fixation of Tissue; Hybridization and Washing; Control Procedures.
In Situ Methods to Localize Transgenes and Transcripts in Interphase Nuclei In situ methods to visualize transgenes (including single copy genes) and their transcripts during interphase from different tissues and plant species. These techniques reduce the time necessary for characterization of transgene integration by eliminating the need for time-consuming segregation analysis, and extend characterization to the interphase nucleus, thus increasing the likelihood of accurate prediction of transgene activity.
Multiple-Target DNA In Situ Hybridization with Enzyme-Based Cytochemical Detection Systems Protocol Protocol for multiple-target DNA in situ hybridization with enzyme-based cytochemical detection systems. Includes: Cell preparations; Cell processing; Probe preparation; Multiple-target in situ hybridization (ISH); ISH with separate probe and target denaturation [for probes with repetitive (e.g., Alu) elements]; Post-hybridization washes; Enzyme-based cytochemical detection; etc..
Nucleic Acid Hybridization General Aspects Discusses the effects of various components of the hybridization solution on the rate of renaturation and thermal stability of DNA hybrids free in solution. Includes: The main parameters that influence hybridization; Additional hybridization variables; Competition in situ hybridization; Oligonucleotide hybridization; Standard in situ hybridization conditions.
Reverse Transcriptase In Situ PCR Protocol This protocol describes a method for reverse transcriptase (RT) in situ PCR. In situ PCR differs from PCR in situ hybridization in the inclusion of a reporter molecule in the amplification step. The two steps of RT in situ PCR that differ from in situ PCR are overnight digestion in RNase-free DNase that is performed after protease digestion, and an RT step, prior to in situ PCR.
Transcript In Situ Hybridization of Whole-Mount Embryos for Phenotype Analysis of RNAi-Treated This protocol fixes and prepares embryos for in situ hybridization to visualize transcript expression patterns. It is a modification of the method developed by Tautz and Pfeifle for whole-mount in situ analysis of embryos. Use of the standard hybridization protocol on RNAi-treated embryos results in high background staining, which makes visualization of transcript expression patterns practically impossible. The following modifications eliminate this problem and allow visualization of transcript.
