20.109(F16):Data analysis (Day7)

Introduction

Protocols

Part 1: Visualize H2AX assay results

The teaching faculty will replace the primary antibody solution with the diluted secondary antibody solution, Alexa Fluor 488 goat anti-mouse diluted 1:200 in blocking solution. Following the Business proposal presentations, you will visualize the results of your H2AX assay according to the below steps.

1. Aspirate the secondary antibody solution.
2. To wash the coverslips, add ~500 μL of TBS per well and rock the plate back and forth, then aspirate.
   • Repeat this step a total of two times.
3. Add 500 μL TBS per well.
4. Obtain a fine gauge (26 3/8) needle, a pair of tweezers, and four glass microscope slides from the front laboratory bench.
   • Carefully press the tip of the needle against the benchtop to bend it into a right angle such that the beveled side of the needle is the interior angle.
5. Use the 'hook' created in Step #4 to lift the coverslip from the bottom of the well, then use the tweezers to 'catch' the coverslip.
   • In total, you only need to visualize two coverslips from the gamma-irradiated plate and two coverslips from the control plate. Therefore, you have several wells that can be used as practice.
6. When you are confident with your ability to retrieve the coverslips from the wells, add 5 μL of mounting media to a glass microscope slide.
7. Place the coverslip cell-side down on the mounting media on the microscope slide.
   • The cell-side of the coverslide is the side that was facing up in the well of the plate.
8. Complete Steps #5-7 for coverslips from two gamma-irradiated and two control wells.
9. Alert the teaching faculty when all four microscope slides are ready and you will be escorted to the microscope in the Engelward laboratory.

Reagents

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