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 ( approximately 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.
Xie
Sheila Q
SQ
MRC Clinical Sciences Centre, Imperial College School of Medicine, London, UK.
Lavitas
Liron-Mark
LM
Pombo
Ana
A
D000483:Alleles; D000818:Animals; D000941:Antigens / Q000276:immunology; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002875:Chromosomes / Q000235:genetics / Q000378:metabolism; D016616:Cryoultramicrotomy / Q000379:methods*; D006801:Humans; D007118:Immunoassay; D007075:Immunoglobulin M / Q000276:immunology; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D010766:Phosphorylation; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism; D016610:Tissue Embedding; D016707:Tissue Fixation
D000941:Antigens; D007075:Immunoglobulin M; D012319:RNA Polymerase II