Improved immunodetection of nuclear antigens after sodium dodecyl sulfate treatment of formaldehyde-fixed cells.
Immunostaining techniques are commonly employed to determine the temporal and spatial patterns of cellular components in in situ preparations of cells and tissues. Usually, cells are formalin-fixed, permeabilized with nonionic detergents, and probed with specific antibodies. The incorporation of a sodium dodecyl sulfate (SDS) treatment after chemical crosslinking has been shown to improve the immunodetection of some cytosolic and cell surface antigens. By incorporating an SDS treatment after crosslinking, we report a significant improvement in the detection of two nuclear antigens (i.e.) the DNA binding proteins apurinic/apyrimidinic endonuclease and DNA polymerase-beta) and bromodeoxyuridine-tagged DNA by indirect immunofluorescence of whole cells. In bromodeoxyuridine-tagged DNA, the improvement in detection after an SDS treatment was observed only after long incorporation protocols (>48 hr) and, interestingly, it was more pronounced in cultured human foreskin keratinocytes than in bovine aorta endothelial cells. In addition, the SDS treatment proved in these studies to be superior to the standard Triton X-100 permeabilization. SDS thus provides a potential means to visualize previously undetectable or poorly detectable nuclear antigens.
Wilson
D M
DM
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, Massachusetts, USA.
Bianchi
C
C
D000818:Animals; D001973:Bromodeoxyuridine / Q000276:immunology / Q000378:metabolism; D019757:Carbon-Oxygen Lyases / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D002417:Cattle; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D019951:DNA Polymerase beta / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D009687:Nuclear Proteins / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*; D013501:Surface-Active Agents / Q000494:pharmacology*
D009687:Nuclear Proteins; D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate; D019951:DNA Polymerase beta; D043223:Deoxyribonuclease IV (Phage T4-Induced); D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D001973:Bromodeoxyuridine