PMC

Detection of IGF-I in channel catfish (I. punctatus) and rainbow trout (O. mykiss) leukocytes. (a,b) IGF-I mRNA and peptide in I. punctatus T-cell line G14D using (a) PCR and (b) immunocytochemistry. (a) Agarose gels of real-time PCR products confirming the presence of IGF-I mRNA (bands 2–4) at the expected size (73 bp) and reference gene 18S rRNA (bands 5–6) at the expected size (85 bp). Bands 1 and 7: molecular weight marker. (b) The majority of the T-cells show IGF-I immunoreactive material (brown color) within the cytoplasm. Nuclei counterstained with haematoxylin. Bar: 60 µm. (c) Head kidney leukocytes freshly isolated from rainbow trout were adjusted to 1 × 10⁷ cells/mL, incubated with mouse mAb against rainbow trout granulocytes (mAbQ4E; []) visualized with a Texas red-coupled anti-mouse antibody as previously described []. Subsequently, cells were fixed for intracellular labeling for IGF-I using the same antiserum as in (b) followed by a FITC-coupled anti rabbit antibody for double immunofluorescence of IGF-I (green) in phagocytic cells (red, arrow). Note the small cells immunoreactive for IGF-I, which do not express the granulocyte marker (single arrowheads) and the large cell immunoreactive for the granulocyte marker, but without immunoreactivity for IGF-I (double arrow heads). Bar: 30 µm.