Fluorescence in situ hybridization on vibratome sections of plant tissues.
This protocol describes the application of fluorescence in situ hybridization (FISH) to three-dimensionally (3D) preserved tissue sections derived from intact plant structures such as roots or florets. The method is based on the combination of vibratome sectioning with confocal microscopy. The protocol provides an excellent tool to investigate chromosome organization in plant nuclei in all cell types and has been used on tissues of both monocot and dicot plant species. The visualization of 3D well-preserved tissues means that cell types can be confidently identified. For example, meiocytes can be clearly identified at all stages of meiosis and can be imaged in the context of their surrounding maternal tissue. FISH can be used to localize centromeres, telomeres, repetitive regions as well as unique regions, and total genomic DNAs can be used as probes to visualize chromosomes or chromosome segments. The method can be adapted to RNA FISH and can be combined with immunofluorescence labeling. Once the desired plant material is sectioned, which depends on the number of samples, the protocol that we present here can be carried out within 3 d.
Prieto
Pilar
P
John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK. 152prarm@uco.es
Moore
Graham
G
Shaw
Peter
P
D002875:Chromosomes / Q000648:ultrastructure; D006813:Humidity; D017404:In Situ Hybridization, Fluorescence; D007202:Indicators and Reagents; D008540:Meiosis; D018613:Microscopy, Confocal / Q000379:methods; D059828:Plant Cells; D018514:Plant Structures / Q000737:chemistry / Q000235:genetics; D010944:Plants / Q000737:chemistry / Q000235:genetics*
D007202:Indicators and Reagents