Previously published procedures were followed for IF and IF-FISH12 (link). IF for myc-tagged RPA32 or Ctc1 (mouse monoclonal, 9B11 or rabbit monoclonal, 71D10, Cell Signaling Technology), HA-tagged Stn1 (3724, Cell Signaling Technology), endogenous Polα (sc-137021, Santa Cruz), and 53BP1 (612522, BD Biosciences) was carried out using the cytoskeleton extraction protocol14 (link). Intensity measurements of RPA32-myc IF were performed in FIJI as follows: nuclei were identified using thresholding, segmented, and identified as regions of interest. The average image background was then subtracted from the image, and the total raw pixel intensity within each area of interest in the channel of interest was calculated. Rad51 (70-001, Bioacademia), and γH2AX (05636, Millipore) were detected in cells fixed in 3% PFA, and foci showing co-localization of Rad51 with γH2AX were quantified. IF imaging was performed on a Zeiss Axioplan II microscope equipped with a Hamamatsu C4742-95 camera using Volocity software or on a DeltaVision (Applied Precision) equipped with a cooled charge-coupled device camera (DV Elite CMOS Camera), a PlanApo 60× 1.42 NA objective or 100× 1.40 NA objective (Olympus America, Inc.), and SoftWoRx software.
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Immunofluorescence Imaging of DNA Repair Factors
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Other organizations : Rockefeller University, Mount Sinai Hospital, Lunenfeld-Tanenbaum Research Institute
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Variable analysis
independent variables
- Myc-tagged RPA32
- Ctc1 (mouse monoclonal, 9B11 or rabbit monoclonal, 71D10)
- HA-tagged Stn1
- Endogenous Polα
- 53BP1
- Rad51
- γH2AX
- Intensity measurements of RPA32-myc IF
- Foci showing co-localization of Rad51 with γH2AX
- Previously published procedures were followed for IF and IF-FISH
- Cytoskeleton extraction protocol was used
- Cells were fixed in 3% PFA
- Imaging was performed on a Zeiss Axioplan II microscope or a DeltaVision (Applied Precision) microscope
- Not explicitly mentioned
- Not explicitly mentioned
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