I am currently working on whole-mount zebrafish in situ hybridization. The zebrafish larvae I am working on are 5 dpf. The protocol I am following is Bernard Thisse and Christine Thisse. The larvae didn't not treated with PTU, but treated with 1% H2O2 and 1% KOH for depigmentation. Proteinase K 10 ug/ml, 10 mins. The signal appeared at around 2 hrs after adding NBT/BCIP (thermofisher, 1 step); however, the issue I am currently encountering is the larve becoming smaller and shrinking very badly after incubating in NBT/BCIP and it seemed that the larve could not rehydrate it back to the original size after incubating the stop solution. These larvae made me hard to do the dorsal-ventral position.

Was wondering if anyone has had similar issues before, and could you give me some suggestions?

Thanks.