20.109(F20):M1D4

Part 1: Complete staining for gamma-H2AX assay

1. Make sure to have TBS solution available before you start. Aspirate the secondary antibody solution off the coverslip and immediately add 150 μL of TBS. Do not let the coverslips dry out during this process.
2. To complete the post secondary wash, add 150 μL of TBS per coverslip, let incubate at room temperature for 3 min covered, then aspirate.
   • Repeat this step twice.
3. Obtain glass slides from the front laboratory bench and label your slides with all of your experimental information and group name, add one drop of mounting media to the slide.
4. Aspirate the final TBS wash and using tweezers place the coverslip cell-side down on the mounting media "spot" on the microscope slide. Try your best to avoid bubbles by slowly placing the coverslip over the mounting media.
   • The cell-side of the coverslide is the side that was facing up in the staining chamber.
5. Complete Steps #3-4 for coverslips from all of the coverslips you stained.
6. Add one small drop of nail polish to each side of your coverslip to seal it to the glass slide.
7. Alert the teaching faculty when all of your microscope slides are ready and you will be escorted to the microscope in the Engelward laboratory.