Quantifying Protein Levels in Zebrafish Development

Protein lysates were obtained from control versus nephrin morphants and from adriamycin non-exposed versus exposed zebrafish larvae. Blots were incubated with a rabbit polyclonal anti-PACAP antibody (produced in our laboratory, as previously described) [16 (link)], a rabbit monoclonal anti-ceruloplasmin antibody (Dako, Glostrup, Denmark), a goat polyclonal anti-GFP antibody (Rockland Immunochemicals, Gilbertsville, USA) and a rabbit monoclonal anti-β-actin antibody (Cell Signaling Technology, Boston, USA). Secondary anti-rabbit and anti-goat antibodies were from Dako. Blots were stained with Western blotting electrochemiluminescence (ECL) detection reagent (Thermo Scientific, Rockford, USA). Detection of signal and measuring of band intensity were performed with ImageJ software. The band intensities of PACAP, ceruloplasmin, and GFP were corrected for the band intensities of the loading control β-actin [26 (link)] and were expressed as a percentage of the control condition. All Western blots were performed at multiple time points during the first 6 days of development and at least in duplicate.