I can't find the information what the people use as a positive control to see the decrease of the hemoglobin in Zebrafish embryos using o-dianisidine staining.
I have tried e.g. salicylic acid according to the paper:
(Gupta, V. et al. Salicylic Acid Induces Mitochondrial Injury by Inhibiting Ferrochelatase Heme Biosynthesis Activity. Mol. Pharmacol. 84, 824–833 (2013),
but the effect is very weak.
Thanks for the ideas,
Ondrej
Hello, Ondrej.
It would be better to use a more specific probe demonstrating that hemoglobin has been synthesized in target cells. Which is a valuable experimental evidence for presentation. Use a specific antibody for zebrafish from Abcam.
Anti-Hemoglobin subunit beta antibody - N-terminal (ab202386).
Regards.
Dear professor Sang Ho Lee,
thank you very much for your advise.
The protocol for the hemoglobin staining works just fine. The problem is, that I can't find the way how to chemically inhibit the synthesis of hemoglobin.
Does exist some chemical that have an effect on formation of hemoglobin, so I would be able to see the difference between treated and untreated zebrafish embryos ?
The alternative approach would be Crispr-Cas knockout of the genes involved in hemoglobin synthesis. I am looking for a cheaper and more straight-forward method/approach.
Thank you,
Ondrej
Hi, Ondrej.
My apology for that I missed your point. How about to use any antibiotics in the culture of zebrafish embryos? Puromycin is one of them that has been used in protein synthesis for biochemical and cell biological purposes. There is no specific inhibitors that block only the hemoglobin synthesis as you know well. The developing zebrafish embryos have so fast cell cycles. Because this kind of treatments including other heavy metals or hypoxia or something else would cause delayed or impaired embryonic development, leading to defects or teratogenic effects in overall development or disorganized embryonic tissues where temporal developmental signals are so crucial. It is not a straight-forward way using a drug inhibitor, rather complicating interpretation from deformed fry.
Although puromycin is a small molecule, you probably have to remove the chorion. Need tests. Good luck.
We're going through a period of poor o-dia staining - works one day and not the next. Any idea where the variability could be coming from??
We had high variability too. But my results were better when I prepared all chemical fresh before each test. Also I think, I can be due to a bleaching.
We bleached the embryos do get rid of the skin pigment.
Currently, we switched to globin-LCR:eGFP fish, so I gave up the o-dia staining. We are about to purchase these fish, so ask me in 2 months if this approach is better :)
Good luck Patrick !
Ondrej
Thanks for the quick answer! I'd be happy to hear how you find the LCR line. Where are you buying if from? ZIRC?
The friend of mine has friends in University of Maryland. They have these fish. We suppose to have them free of charge.
We should have few breading pairs, so hopefully we will have our own colony soon.
Dr.Adamovsky,
Were you able to find a chemical or any other straightforward alternative, that can work as a positive control, for zebrafish embryo o-dianisidine staining? This would be a very useful tip.
- Badges
- Science topic
- Similar topics
- Engineering
- Modeling