GSBS Student Publications
Title
Immunofluorescence imaging of DNA damage response proteins: optimizing protocols for super-resolution microscopy.
Publication Date
2009-05-01
UMMS Affiliation
Department of Biochemistry and Molecular Pharmacology
Document Type
Article
Disciplines
Life Sciences | Medicine and Health Sciences
Abstract
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.
Keywords
immunofluorescence, antibody purification, RNAi to assess antibody specificity, antibody mixes, antibody cocktails, sequential antibody staining, super-resolution microscopy, 4Pi microscopy
DOI of Published Version
10.1016/j.ymeth.2009.02.009
Source
Methods. 2009 May;48(1):63-71. Epub 2009 Feb 24. Link to article on publisher's website
Journal/Book/Conference Title
Methods (San Diego, Calif.)
Related Resources
PubMed ID
19245833
Repository Citation
Bennett, Brian Thomas; Bewersdorf, Jorg; and Knight, Kendall L., "Immunofluorescence imaging of DNA damage response proteins: optimizing protocols for super-resolution microscopy." (2009). GSBS Student Publications. 1596.
https://escholarship.umassmed.edu/gsbs_sp/1596