Hello everyone,

I am encountering some issues with AFOG staining on my cryosections, and I would greatly appreciate your advice.

I am working with 20 µm thick cryosections of zebrafish tissue that was fixed in 4% PFA, transferred to sucrose, embedded in a cryoprotective medium, and stored at -80°C. The sections were cut on a cryostat and stored at -20°C. Stab injury was inflicted to zebrafish muscles, and the idea was to show injury and regeneration process. I followed a staining protocol as outlined below:

Despite following this protocol, I am encountering two issues shown in attached pictures:

• Inconsistent staining: Half of the sections stain well, but the other half appears shriveled and distorted.
• Non-specific blue background: There is a lot of non-specific blue staining that I can't seem to eliminate.

I have introduced an additional fixation step with 10% formalin as described in a paper by Oudhoff, 2024, Article Skeletal muscle regeneration after extensive cryoinjury of c...

Could anyone provide insight into why only part of my sections stain properly while the other part shrivels? Also, what could be causing the non-specific blue staining?

Any advice or suggestions on how to improve the consistency and reduce the background staining would be greatly appreciated!

Thank you in advance for your help!

Best regards, Jovana