Immunofluorescence imaging of DNA damage response proteins: optimizing protocols for super-resolution microscopy.
Department of Biochemistry and Molecular Pharmacology
Medical Subject Headings
Microscopy, Fluorescence; Antibody Specificity
Life Sciences | Medicine and Health Sciences
Immunofluorescence imaging has provided captivating visual evidence for numerous cellular events, from vesicular trafficking, organelle maturation and cell division to nuclear processes including the appearance of various proteins and chromatin components in distinct foci in response to DNA damaging agents. With the advent of new super-resolution microscope technologies such as 4Pi microscopy, standard immunofluorescence protocols deserve some reevaluation in order to take full advantage of these new technological accomplishments. Here we describe several methodological considerations that will help overcome some of the limitations that may result from the use of currently applied procedures, with particular attention paid to the analysis of possible colocalization of fluorescent signals. We conclude with an example of how application of optimized methods led to a breakthrough in super-resolution imaging of nuclear events occurring in response to DNA damage.
Rights and Permissions
Citation: Methods. 2009 May;48(1):63-71. Epub 2009 Feb 24. Link to article on publisher's website
immunofluorescence, antibody purification, RNAi to assess antibody specificity, antibody mixes, antibody cocktails, sequential antibody staining, super-resolution microscopy, 4Pi microscopy