Hello!
I'm having trouble removing alcian blue specks from my zebrafish larvae stains. See photo for specks of alcian blue in the right corner of ANC.
I'm following the Walker & Kimmel (2007) protocol.
I don't think its from under bleaching.
I think I may not be filtering the stock solution properly or could be a pH issue.
The protocol is an acid free stain. I wonder if using glacial acetic acid might correct this.
In doing so, would I have to remove the magnesium chloride that is used to differentiate the blues as the Kimmel/Walker paper suggests? I really like the blue color I'm receiving. I plan to start trouble shooting next week and was hoping for suggestions or feedback.
Thank you,
Eileen
Are you referring to the blue circles on the center right? Those look like actual staining. It is possible that this is incomplete staining. Based on the location through, it is more likely that it is the primary lamellae of the gills. Looking at the specimen in a lateral orientation would tell you for sure.
Or are you referring to the small specs in the upper right corner? If so, those are not very many particles and shouldn't interfere with any analyses. That said, if you don't like them then you can try filtering your stain before using it. Either push it through a syringe filter, or even easier, pour it through filter paper set into a glass funnel.
Hope that helps. Best of luck.
- Melissa
No, I am not. I am referring to the specks in the corner of the specimen. I tried a low and high acid stain and I think the specks are caused by unspecific staining or an artifact. They may suppose to be there due to pH.
If you think that it may be due to acidity, then you can try the acid-free method for this stain. See here: Walker MB, Kimmel CB. 2007. A two-color acid-free cartilage and bone stain for zebrafish larvae. Biotech Histochem 82:23–28.
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