Authentication of a novel antibody to zebrafish collagen type XI alpha 1 chain (Col11a1a)

Abstract

Objective Extracellular matrix proteins play important roles in embryonic development and antibodies that specifically detect these proteins are essential to understanding their function. The zebrafish embryo is a popular model for vertebrate development but suffers from a dearth of authenticated antibody reagents for research. Here, we describe a novel antibody designed to detect the minor fibrillar collagen chain Col11a1a in zebrafish (AB strain). Results The Col11a1a antibody was raised in rabbit against a peptide comprising a unique sequence within the zebrafish Col11a1a gene product. The antibody was affinity-purified and characterized by ELISA. The antibody is effective for immunoblot and immunohistochemistry applications. Protein bands identified by immunoblot were confirmed by mass spectrometry and sensitivity to collagenase. Col11a1a knockout zebrafish were used to confirm specificity of the antibody. The Col11a1a antibody labeled cartilaginous structures within the developing jaw, consistent with previously characterized Col11a1 antibodies in other species. Col11a1a within formalin-fixed paraffin-embedded zebrafish were recognized by the antibody. The antibodies and the approaches described here will help to address the lack of well-defined antibody reagents in zebrafish research.

The Shape of the Jaw—Zebrafish Col11a1a Regulates Meckel’s Cartilage Morphogenesis and Mineralization

“…In our previous studies, zebrafish col11a1a expression was located within the developing jaw using in situ hybridization [ 24 , 25 ]. Using a recently developed antibody, we also identified the zebrafish Col11a1a protein within the developing jaw [ 26 ]. Based on previous findings, we investigated the effect of col11a1a gene knockdown on the cellular organization of chondrocytes within Meckel’s cartilage and the timing and location of mineralization of the developing jaw of zebrafish, both events that play critical roles in determining the shape of the jaw.…”