Hi all,
I am treating my 8 day old Arabidopsis thaliana saplings with different concentrations of H2O2 for variable time periods. After the treatment I want to check the viability of cells (specially roots cells). I looked for various viability stains and I found FAD to be the best one (please suggest me if you think any other stain better than FAD).
Because I am treating my plant saplings with H2O2 for 6 Hrs, I am worried if it will react or affect FAD during staining procedure.
Please let me know what are your opinions/suggestions.
Thank you very much!
I suggest you do Propidium Iodide (PI) staining of your root samples (treated vs control). PI staining is cheap, very fast, and easy. PI is a membrane integrity marker. PI cannot enter viable/living cells, hence the fluorescence is only seen in cell wall/membrane whereas it can enter inside dead cells (with damaged/compromised cell surface integrity)and usually stains the interior of cells especially nucleus/DNA. Note that Propidium iodide and ethidium bromide are chemically/functionally very similar (DNA binding/intercalating agents). Hope it helps you.
Hi Avinash,
I'm not familiar with FAD but we generally use Evan's Blue staining to visualize cell viability/integrity in roots. It is very easy and clear, and you only need a regular light microscope. Currently we also do H2O2 tolerance assays and it also seems to work with that.
Short protocol:
"Evans blue staining for cell viability in root tips
Arabidopsis seedlings were taken for root cell integrity analysis by Evans blue staining after air and ethylene pre-treatments followed by both hypoxia and post-hypoxia time-points. Seedlings were incubated in 0.25% aqueous Evans blue staining solution for 15 min in the dark, subsequently washed three times with Milli-Q water to remove excess dye and finally imaged using light microscopy (OLYMPUS BX50WI, 10x objective)."
From: https://www.nature.com/articles/s41467-019-12045-4#Sec8
Results are visible in Supplementary Figure 3.
Let me know if this is helpful :)
Sjon
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining