Are the results you have already "not as expected" because:

• the probe test did not work, or because
• the results are not similar to previous experiments?

If the probe didn't work, what have you already tried/what failed/etc? At the moment, we don't really know what to suggest, because you haven't really explained what did or didn't work.

If the results aren't as expected, is the issue with no response/over expression/ seemingly random values? Are you measuring anything within the working range of the test? If you can only measure from 1-10 arbitrary units and the roots are yielding 0.01-0.1 units, then you're essentially measuring blanks. If the roots are yielding 1000 units, then the signal is exceeding the capacity of the test and you are only measuring the maximum of the test.

Have you tested the method with a blank (eg water) and a control sample (dilute H2O2) to check it actually works the way it is supposed to?

Thank you Matthew Wheal. The main issue I am encountering is that the results from the treatment group are showing low fluorescence intensity than expected. The probe test itself appears to be working, as I’ve tested it with a control sample, and it shows normal fluorescence. However, in the treatment group, the fluorescence intensity is significantly lower than both the control and previous experiments.

If the intensity has declined, are you using the same stock solution of DCFH-DA? Could it have degraded since the original tests you ran? Perhaps check how long it stays effective and prepare a new stock each time. I don't know what form it is (powder, crystals, liquid) but it could well go stale if left exposed to air and light and heat.

Could pH or other environmental circumstances alter the degree of response you are seeing in the latest set of root samples?