Whole-mount Immunostaining of Ubap2 in Zebrafish

Whole-mount immunostaining of Ubap2 in the zebrafish larvae was performed as previously described^{58 (link)}. Briefly, 3 dpf larvae were fixed in 4% paraformaldehyde diluted in 1 × PBS with 0.25% Triton X (PBST) at 4 °C overnight. After PBST washing, antigens retrieval was performed using TrisHCl (pH 9.0, 150 mM) at 70 °C for 20 min. The larvae were washed with PBST and then treated with 0.05% trypsin-EDTA for 45 min on ice, and incubated with a blocking buffer (2% normal goat serum, 1% bovine serum albumin, and 1% dimethyl sulfoxide) for 1 h. The larvae were incubated overnight at 4 °C with the primary antibody (Ubap2, 1:10, Abcepta, AP12773a) or without the primary antibody as a negative control and then incubated with a secondary antibody conjugated with Alexa fluorophore 488 (1:500, Invitrogen). Larvae were mounted in 1% low melting agarose and imaged with a confocal microscope (FV1000 confocal microscope, Olympus)^{58 (link)}.