Dears,
I am trying to stain zebrafish leave with alizarin red Bone staining, to stain invertebral bone specifically.
I have tried to follow four different protocol with no specific stain, one of them only show the notochord with a faint stain.
I have tried short fixation, add triton-x to enhance larvae permeability, increase alizarin concentration as mentioned on different protocols.
Please any suggestions? And if anyone works with same age at 5dpf for bone staining
Referring to classic developmental table of ossification in zebrafish, there are only vertebral centra at 5 dpf and virtually no other calcified elements. So you may be expecting too much, where there is actually little or nothing there:
Article Developmental Morphology of the Axial Skeleton of the Zebraf...
First use a larger fish (10-14 days) ), to be sure your bone stain is working. Then use the same protocol on younger specimens. Good luck with your experiment.
Early stages of larvae are mostly cartilage. If your larvae are this young, that is probably why you do not see alizarin staining for bone. I suggest you co-stain with alcian blue (cartilage stain) and alizarin red.
If your interest is in quantifying the amount of bone present at certain ages, then you can still do that even with a co-stin. Our lab has published methods for how to do that using ImageJ:
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For co-staining, these two protocols are good:
- https://wiki.zfin.org/display/prot/A+Simple+Whole-Mount+Staining+Protocol+For+Bone+And+Or+Cartilage+In+Adults+And+Larvae
- https://wiki.zfin.org/display/prot/Clearing+And+Staining+For+Larval+Fish+Cartilage+And+Bone
I also recommend this protocol, which I have had very good success with in both medaka and zebrafish larvae. I find that staining with alcian blue first and alizarin red second works well for me, but either should work for you. Note, that in this protocol I use the Alizarin Red I solution in the Method 2, step 3 instead of the Alizarin Red II solution for better results.
- https://shigen.nig.ac.jp/medaka/medakabook/index.php?4.4.1%20Staining%20of%20cartilage%20and%20bone%20in%20whole%20embryos%2C%20larvae%20and%20adults%20by%20Alcian%20blue%20and%20Alizarin%20red
Regardless of the protocol you choose, be careful in two of the steps:
- The enzyme digestion step. Make sure you make the enzyme fresh each time you use it. Don't leave the larvae too long in the trypsin. I would recommend a minimum of 30 min but not more than 1 hour or you will lose the eyes and skin. If you start to see the eyes coming loose then take it out.
- The bleaching (H2O2 and KOH) of soft tissues. This can also go too far. After 20-30 min, check on the specimens. If the eyes are transparent and the skin pigment is a light brown (or gone) then they are done. If not, let them go a longer, checking on them with a microscope every 5-10 min.
I hope that helps and good luck.
- Melissa
Thank you so much Elizabeth and melissa for your valuable feedback, really appreciate
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