20-25 larvae (96h) were randomly selected, washed 2-3 times with PBS, and then stained with AO(5ug/ml) and DCFH-DA(10uM) stain for 30 min separately and then washed with PBS 2-3 times to remove the extra stain. But the larvae after staining becomes somewhat sticky and when observed under the fluorescent microscope, they seem to be somewhat degraded. Can any one suggest me something regarding the above-mentioned problem? Thank you.
Are you anesthetizing or euthanizing the larvae? If the latter, then the tissue may be starting to break down/decompose during the staining time. You should be able to do AO staining and DCFH-DA staining in vivo; where you will need to use embryo medium and not PBS. Also, are you staining the same fish with both stains? All of our protocols and papers I've read stain one set of larvae with AO and a separate set of larvae with DCFH-DA.
- Melissa
Dear mam
I didn't anesthetize the larvae, I just washed them with PBS (2-3 times) and stained one set of larvae with AO and a separate set of larvae with DDFH-DA. And now I will try the things that you recommended. Thank you for the suggestions mam.
Hello Shiv Kumar
Your methodology seems quite similar to what we do in our lab. But can you let me know about the solvent used to dilute the DCFDA / H2DCFDA kit? Try to Use solvent which known non-toxic to zebrafish and also at lower concentrations. Alternatively you can also reduce the incubation time and working concentration of DCFDA / H2DCFDA based on the fluorescence you saw in the previous trials.
All the best.
Hello Sir,
I am using PBS as a solvent to dilute the DCFDA(10uM) dye. Can I expose the larvae to DCFDA in E3 media because no toxic effect will be there?
The incubation time can be reduced by up to 15 min.
Thank you for your valuable information.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line