CryoFISH: Fluorescence In Situ Hybridization on Ultrathin Cryosections
The visualization of cellular structures and components has become an invaluable tool in biological and medical sciences. Imaging subcellular compartments and single molecules within a cell has prompted the development of a wide range of sample preparation techniques as well as various microscope devices to obtain images with increased spatial resolution. Here, we present cryoFISH, a method for fluorescence in situ hybridization (FISH) on thin (∼150 nm thick) cryosections from sucrose-embedded fixed cells or tissues. CryoFISH can be used in combination with immunodetection (IF) of other cellular components. The main advantages of cryoFISH and cryoIF over whole-cell labeling methods are increased spatial resolution with confocal microscopy, greater sensitivity of detection due to increased probe accessibility, and better image contrast. CryoFISH and cryoIF methods typically used on samples fixed in conditions that preserve ultrastructure, are compatible with the labeling of cells in their tissue context and are ideal for correlative studies that compare fluorescence with electron microscopy.
Key wordsImaging Immunofluorescence Fluorescence in-situ hybridization Interchromosomal interactions Tokuyasu cryosections RNA polymerase II
We thank Michael Hollinshead, Kate Liddiard, and Miguel R. Branco for their contribution to the development and application of cryoFISH approaches, and Miguel R. Branco and Tiago Branco for their contributions to the development of macros for quantitative analyses of cryoFISH images. We thank the Medical Research Council (UK) for funding.
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