Immunofluorescence staining protocol to detect ssDNA (CldU) and active replication (EdU)

Immunofluorescence staining protocol to detect ssDNA (CldU) and active replication (EdU)

1.     Seed around 0.5X10⁵ or 1X10⁵ cells in a 6-well plate with coverslips. (The number of cells depends on the cell morphology and size. Ensure that the cells are around 60-70% confluent on the day of drug treatment).

2.     While seeding the cells ensure that the growth media is supplemented with 50uM CldU (or nucleotide analog of your choice) for 48hrs to ensure that the parental DNA strands are well coated with the analog during replication.

3.     While incubating the 6-well plate, ensure to wrap it with a foil to protect from direct contact with light.

4.     After 48hrs incubation, remove the media from the wells and gently wash (swirl) the wells three times with 1X phosphate-buffered saline (PBS) to wash off any residual CldU.

5.     Now treat the cells/wells according to your requirement. For control – Treat the cells with just regular media with 10uM EdU for 30mins and 2hrs respectively. For the experimental set up, treat the cells with 10uM EdU + Hydroxyurea (dose range – 0.25mM to 2mM) for 2hrs. (DO NOT INCLUDE CldU at this step). Incubate the plate for the desired duration of time (2hrs) and wrap it with a foil to protect from direct contact with light.

6.     After the desired incubation, remove the media from the wells and gently wash (swirl) the wells three times with 1X PBS to wash off any residual EdU and/or hydroxyurea.

7.     Treat the cells with 0.5% Triton X-100 made in PBS on ice for exactly 5mins. Do not rock or shake the plate and cover the plate with a foil to protect it from light.

8.     Repeat the washing step with 1X PBS for three times to remove any traces of 0.5% Triton X-100. Just swirl and do not shake the plate.

9.     Cells are then fixed with 4% Formalin for 15mins at room temperature (RT).

10.  Repeat the washing step with 1X PBS for three times to remove the fixative. Make sure that the plate/wells are washed well. (If you desire to stop the protocol, then cover the wells with 1X PBS and store the plate, wrapped in a foil at 4°C.

11.  Treat the cells/well with 3% Bovine serum albumin (BSA) made in PBS + 0.1% Triton-X 100 (blocking solution) for 30mins at RT.

12.  Repeat the washing step with 1X PBS for three times to remove excess BSA.

13.  Fixed cells are then incubated with primary antibodies (Abs) against CldU (Abcam ab6328 1:100; Rat Monoclonal anti-BrdU which is also specific for CldU) made in the blocking solution at 37°C for 1hr. Make sure that the plate is covered with a foil and protected from light.

14.  Repeat the washing step with 1X PBS for three times to remove excess primary Abs.

15.  The cells are then treated with secondary Abs (Alexa 594 1:200; Goat anti-rat) made in the blocking solution at RT for 1hr. Make sure that the plate is covered with a foil and protected from light.

16.  Repeat the washing step with 1X PBS for three times to remove excess secondary Abs.

17.  For EdU staining, staining was carried out with Click-iT EdU imaging kit (Alexa 488) by Invitrogen. (NO NEED to again fix or do pre-extraction) . The cells can be simply stained using the EdU click-it staining solution as mentioned and described on page 7 of the Click-iT EdU imaging kit online protocol (https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FClick_iT_EdU_Flow_Cytometry_Assay_Kits_100_tests_PI.pdf&title=Q2xpY2staVQgRWRVIEZsb3cgQ3l0b21ldHJ5IEFzc2F5IEtpdHMgKjEwMCB0ZXN0cyo=)

18.  After EdU staining, cells are washed again with 1X PBS for three times and the coverslips mounted onto glass slides using Vectashield mounting medium containing DAPI (Vector Laboratories).

NOTE: If you only wish to stain the cells for CldU (ssDNA) and not for EdU, then please avoid the EdU staining part (step-17) and when you treat the cells with the drug (step-5, do not add EdU in the media, rest all will remain the same).

Category