Recycling cultured cells for immunofluorescent labeling.
A method to use sequential rounds of immunofluorescent labeling in cell cultures is presented. The method is based on the utilization of a non-liquid reducing agent, sodium dithionite, in conjunction with ionic or non-ionic detergents (SDS or TX100, respectively) at room temperature. This method preserves cell morphology and substrate antigenicity, and operates through the complete extraction of most primary and secondary antibodies. Using this protocol, the sequential immunolocalization of different proteins is possible, without signal interference with previous immunolabeling rounds. In addition, the method is also useful to recycle blotted membranes in immunoblots.
Espada
J
J
Departamento de Bioquímica, Instituto de Investigaciones Biomédicas, CSIC, Facultad de Medicina, Universidad Autónoma de Madrid, Arturo Duperier 4, 28029 Madrid, Spain. jespada@iib.uam.es
Juarranz
A
A
Villanueva
A
A
Cañete
M
M
Andrés
I
I
Stockert
J C
JC
D000818:Animals; D002478:Cells, Cultured; D003247:Conservation of Natural Resources; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis*; D006367:HeLa Cells / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D051379:Mice; D019163:Reducing Agents / Q000032:analysis / Q000737:chemistry; D015203:Reproducibility of Results; D012967:Sodium Dodecyl Sulfate / Q000032:analysis / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D013431:Sulfates / Q000032:analysis / Q000737:chemistry; D013501:Surface-Active Agents / Q000032:analysis / Q000737:chemistry
D005456:Fluorescent Dyes; D019163:Reducing Agents; D013431:Sulfates; D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate; C004850:sodium dithionate