PubMed · 7705180
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T Raap. 1995-01-01. Cytometry for CGH.. https://doi.org/10.1002/cyto.990190102
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When studying the localization of RNA within a cell, it is important to probe RNA relative to a subcellular landmark, such as a gene locus (transcription site). In this chapter, we describe a single-molecule fluorescence in situ hybridization (smFISH) protocol for labeling target RNAs and their DNA loci in bacteria. This is a versatile protocol applicable to various genes and species, providing valuable insights into RNA localization in bacterial cells.
Immunohistostaining (IHS) is a widely used technique in diagnostic and research laboratories in which specific antibodies are used to detect and visualize a protein of interest in cells or tissues. Similarly, with specific oligonucleotide probes, the fluorescence in situ hybridization (FISH) method allows one to visualize genomic regions and to analyze its localization in the nuclear space. Here, we describe a combined FISH-IHS technique that enables researchers to determine the localization of protein and genomic loci in plant nuclei simultaneously. This method can be applied to extracted nuclei and sections of paraffin-embedded tissues. It provides a valuable tool to improve our understanding of nuclear dynamics by revealing the spatial relationship between specific genomic loci and target proteins.
The chromosome constitution on H96276-2, which is a stable line derived from a sterile wheat-Agropyron intermedium partial amphiploid Zhong 3 cross to common wheat Hui 75, was analyzed by sequential GISH and FISH. The results of GISH using genomic DNA of Agropyron intermedium as probe showed that H96276-2 possessed 20 pairs of wheat chromosomes and one pair of translocation chromosomes of wheat and Ag. intermedium. The translocated alien chromosome fragments located on the terminals of one pair of wheat chromosomes. Furthermore, reprobing of repeat sequence pSc119 was used to identify the wheat chromosomes involved in the translocation. It was determined that the alien fragments located in the terminals of the short arms of 2B chromosomes of wheat.