Retina-derived fetuin (RDF): Analysis by immunocytochemistry, reverse transcriptase-polymerase chain reaction and Southern blot

Purpose. This study was undertaken to determine the presence of retina-deri ved fetuin (RDF) protein and its message in retinal tissues and retinal pig ment epithelial (RPE) cells. The techniques utilized in this study included light microscopy, immunochemistry, Western blot, reverse transcriptase-pol ymerase chain reaction (RT-PCR) and Southern blot. Methods. Retinal tissues and sections from embryonic, early postnatal and a dult normal rats and retinal pigment epithelial (RPE) cells from postnatal rats were immunostained for fetuin with a polyclonal fetuin antibody and a peroxidase conjugated-secondary antibody using immunocytochemistry and West ern blot analyses. The cDNA generated from RNA isolated from early postnata l rat retinas and RPE was probed with primers for rat fetuin, amplified by PCR and the PCR products were analyzed by Southern blot. Results. Fetuin (RDF) was immunolocalized to cells of the neuroepithelium i n retinas of early postnatal rats and most prominently in the nuclei and pe rinuclear region of cultured neonatal rat RPE cells. In adult retinas, gang lion cells, inner segments of photoreceptor cells, some components of the o uter plexiform layer, ganglion cells and optic nerve processes were immunor eactive for the fetuin protein. As shown by Western blot, fetuin (RDF) was higher in embryonic and early postnatal retinas than in late postnatal reti nas, indicating that this protein may be developmentally regulated. Using R T-PCR, the message for rat fetuin was demonstrated in the retina and RPE of normal postnatal rats. Southern blot confirmed that the PCR product from t he retina and RPE was generated from rat fetuin mRNA as well as from rat li ver, the primary source of fetuin. Conclusions. Fetuin, termed retina-derived fetuin (RDF), is reported for th e first time in retinal tissues. Fetuin is a cysteine protease inhibitor th at may play a role in support of neuronal cell survival during early retina l development and the maintenance of neuronal activity. RDF may interact wi th other growth factors and cytokines in providing trophic support for neur ons and possibly other cells of the developing retina.