Hello,

I am trying to use transverse cryosections of larval zebrafish tails for ISH and histological staining, but my sections often all off my slide (occasionally, one or two stay on). How I process my tissue: The tissue is fixed in 4% PFA overnight, then washed in 1X PBS and stored in MeOH until embedding. I decalcify the tissue in 0.5M EDTA and prep the tissue overnight in sucrose. I embed the tissue in OCT/sucrose, flash freeze in isopentane, and store at -80C at least overnight.

I then use the cryostat as normal, and I make 16um sections. I check that I have tissue as I'm stamping them on my Superfrost slides. I leave them at room temp for at least an hour, then store them at -20C until use.

Before I start my staining or ISH, I thaw my slides for at least 1h (often more) at 60C. I perform my stain/ISH in coplin jars, and I'm careful with how I pour solutions in and out. Notably, the protocols work for other types of sections, so I'm wondering if it's my tissue.

I heard that cartilage sections can be finicky, and my tail sections have some of the fin cartilages, but I didn't think it'd be such a big problem. Does anyone have any experience with zebrafish tail sections or otherwise slippery sections?