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

Harold J. Sheedlo, Raghu Krishnamoorthy, Tammy Nelson, Neeraj Agarwal, Jingjing Liu, Rouel S. Roque, Robert J. Wordinger, C. David Jaynes, Anne Marie Brun-Zinkelnagel, Paul O'Brian, John E. Aschenbrenner, James E. Turner

Research output: Contribution to journalArticle

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Abstract

Purpose. This study was undertaken to determine the presence of retina-derived fetuin (RDF) protein and its message in retinal tissues and retinal pigment epithelial (RPE) cells. The techniques utilized in this study included light microscopy, immunochemistry, Western blot, reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot. Methods. Retinal tissues and sections from embryonic, early postnatal and adult 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 Western blot analyses. The cDNA generated from RNA isolated from early postnatal 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 in retinas of early postnatal rats and most prominently in the nuclei and perinuclear region of cultured neonatal rat RPE cells. In adult retinas, ganglion cells, inner segments of photoreceptor cells, some components of the outer plexiform layer, ganglion cells and optic nerve processes were immunoreactive for the fetuin protein. As shown by Western blot, fetuin (RDF) was higher in embryonic and early postnatal retinas than in late postnatal retinas, indicating that this protein may be developmentally regulated. Using RT-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 the retina and RPE was generated from rat fetuin mRNA as well as from rat liver, the primary source of fetuin. Conclusions. Fetuin, termed retina-derived fetuin (RDF), is reported for the first time in retinal tissues. Fetuin is a cysteine protease inhibitor that may play a role in support of neuronal cell survival during early retinal development and the maintenance of neuronal activity. RDF may interact with other growth factors and cytokines in providing trophic support for neurons and possibly other cells of the developing retina.

Original languageEnglish
Pages (from-to)465-471
Number of pages7
JournalCurrent Eye Research
Volume19
Issue number6
DOIs
StatePublished - 1 Jan 1999

Fingerprint

Fetuins
Southern Blotting
Reverse Transcriptase Polymerase Chain Reaction
Retina
Immunohistochemistry
Retinal Pigments
Western Blotting
Epithelial Cells
Ganglia
Polymerase Chain Reaction
Cysteine Proteinase Inhibitors
Immunochemistry
Photoreceptor Cells
Proteins
Antibodies

Keywords

  • Fetuin
  • Optic nerve
  • Protease inhibitor
  • Rat
  • Retina
  • Retinal ganglion cell
  • Retinal pigment epithelium
  • Western blot

Cite this

Sheedlo, Harold J. ; Krishnamoorthy, Raghu ; Nelson, Tammy ; Agarwal, Neeraj ; Liu, Jingjing ; Roque, Rouel S. ; Wordinger, Robert J. ; Jaynes, C. David ; Brun-Zinkelnagel, Anne Marie ; O'Brian, Paul ; Aschenbrenner, John E. ; Turner, James E. / Retina-derived fetuin (RDF) : Analysis by immunocytochemistry, reverse transcriptase-polymerase chain reaction and Southern blot. In: Current Eye Research. 1999 ; Vol. 19, No. 6. pp. 465-471.
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title = "Retina-derived fetuin (RDF): Analysis by immunocytochemistry, reverse transcriptase-polymerase chain reaction and Southern blot",
abstract = "Purpose. This study was undertaken to determine the presence of retina-derived fetuin (RDF) protein and its message in retinal tissues and retinal pigment epithelial (RPE) cells. The techniques utilized in this study included light microscopy, immunochemistry, Western blot, reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot. Methods. Retinal tissues and sections from embryonic, early postnatal and adult 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 Western blot analyses. The cDNA generated from RNA isolated from early postnatal 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 in retinas of early postnatal rats and most prominently in the nuclei and perinuclear region of cultured neonatal rat RPE cells. In adult retinas, ganglion cells, inner segments of photoreceptor cells, some components of the outer plexiform layer, ganglion cells and optic nerve processes were immunoreactive for the fetuin protein. As shown by Western blot, fetuin (RDF) was higher in embryonic and early postnatal retinas than in late postnatal retinas, indicating that this protein may be developmentally regulated. Using RT-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 the retina and RPE was generated from rat fetuin mRNA as well as from rat liver, the primary source of fetuin. Conclusions. Fetuin, termed retina-derived fetuin (RDF), is reported for the first time in retinal tissues. Fetuin is a cysteine protease inhibitor that may play a role in support of neuronal cell survival during early retinal development and the maintenance of neuronal activity. RDF may interact with other growth factors and cytokines in providing trophic support for neurons and possibly other cells of the developing retina.",
keywords = "Fetuin, Optic nerve, Protease inhibitor, Rat, Retina, Retinal ganglion cell, Retinal pigment epithelium, Western blot",
author = "Sheedlo, {Harold J.} and Raghu Krishnamoorthy and Tammy Nelson and Neeraj Agarwal and Jingjing Liu and Roque, {Rouel S.} and Wordinger, {Robert J.} and Jaynes, {C. David} and Brun-Zinkelnagel, {Anne Marie} and Paul O'Brian and Aschenbrenner, {John E.} and Turner, {James E.}",
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Sheedlo, HJ, Krishnamoorthy, R, Nelson, T, Agarwal, N, Liu, J, Roque, RS, Wordinger, RJ, Jaynes, CD, Brun-Zinkelnagel, AM, O'Brian, P, Aschenbrenner, JE & Turner, JE 1999, 'Retina-derived fetuin (RDF): Analysis by immunocytochemistry, reverse transcriptase-polymerase chain reaction and Southern blot', Current Eye Research, vol. 19, no. 6, pp. 465-471. https://doi.org/10.1076/ceyr.19.6.465.5274

Retina-derived fetuin (RDF) : Analysis by immunocytochemistry, reverse transcriptase-polymerase chain reaction and Southern blot. / Sheedlo, Harold J.; Krishnamoorthy, Raghu; Nelson, Tammy; Agarwal, Neeraj; Liu, Jingjing; Roque, Rouel S.; Wordinger, Robert J.; Jaynes, C. David; Brun-Zinkelnagel, Anne Marie; O'Brian, Paul; Aschenbrenner, John E.; Turner, James E.

In: Current Eye Research, Vol. 19, No. 6, 01.01.1999, p. 465-471.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Retina-derived fetuin (RDF)

T2 - Analysis by immunocytochemistry, reverse transcriptase-polymerase chain reaction and Southern blot

AU - Sheedlo, Harold J.

AU - Krishnamoorthy, Raghu

AU - Nelson, Tammy

AU - Agarwal, Neeraj

AU - Liu, Jingjing

AU - Roque, Rouel S.

AU - Wordinger, Robert J.

AU - Jaynes, C. David

AU - Brun-Zinkelnagel, Anne Marie

AU - O'Brian, Paul

AU - Aschenbrenner, John E.

AU - Turner, James E.

PY - 1999/1/1

Y1 - 1999/1/1

N2 - Purpose. This study was undertaken to determine the presence of retina-derived fetuin (RDF) protein and its message in retinal tissues and retinal pigment epithelial (RPE) cells. The techniques utilized in this study included light microscopy, immunochemistry, Western blot, reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot. Methods. Retinal tissues and sections from embryonic, early postnatal and adult 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 Western blot analyses. The cDNA generated from RNA isolated from early postnatal 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 in retinas of early postnatal rats and most prominently in the nuclei and perinuclear region of cultured neonatal rat RPE cells. In adult retinas, ganglion cells, inner segments of photoreceptor cells, some components of the outer plexiform layer, ganglion cells and optic nerve processes were immunoreactive for the fetuin protein. As shown by Western blot, fetuin (RDF) was higher in embryonic and early postnatal retinas than in late postnatal retinas, indicating that this protein may be developmentally regulated. Using RT-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 the retina and RPE was generated from rat fetuin mRNA as well as from rat liver, the primary source of fetuin. Conclusions. Fetuin, termed retina-derived fetuin (RDF), is reported for the first time in retinal tissues. Fetuin is a cysteine protease inhibitor that may play a role in support of neuronal cell survival during early retinal development and the maintenance of neuronal activity. RDF may interact with other growth factors and cytokines in providing trophic support for neurons and possibly other cells of the developing retina.

AB - Purpose. This study was undertaken to determine the presence of retina-derived fetuin (RDF) protein and its message in retinal tissues and retinal pigment epithelial (RPE) cells. The techniques utilized in this study included light microscopy, immunochemistry, Western blot, reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot. Methods. Retinal tissues and sections from embryonic, early postnatal and adult 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 Western blot analyses. The cDNA generated from RNA isolated from early postnatal 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 in retinas of early postnatal rats and most prominently in the nuclei and perinuclear region of cultured neonatal rat RPE cells. In adult retinas, ganglion cells, inner segments of photoreceptor cells, some components of the outer plexiform layer, ganglion cells and optic nerve processes were immunoreactive for the fetuin protein. As shown by Western blot, fetuin (RDF) was higher in embryonic and early postnatal retinas than in late postnatal retinas, indicating that this protein may be developmentally regulated. Using RT-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 the retina and RPE was generated from rat fetuin mRNA as well as from rat liver, the primary source of fetuin. Conclusions. Fetuin, termed retina-derived fetuin (RDF), is reported for the first time in retinal tissues. Fetuin is a cysteine protease inhibitor that may play a role in support of neuronal cell survival during early retinal development and the maintenance of neuronal activity. RDF may interact with other growth factors and cytokines in providing trophic support for neurons and possibly other cells of the developing retina.

KW - Fetuin

KW - Optic nerve

KW - Protease inhibitor

KW - Rat

KW - Retina

KW - Retinal ganglion cell

KW - Retinal pigment epithelium

KW - Western blot

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U2 - 10.1076/ceyr.19.6.465.5274

DO - 10.1076/ceyr.19.6.465.5274

M3 - Article

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VL - 19

SP - 465

EP - 471

JO - Current Eye Research

JF - Current Eye Research

SN - 0271-3683

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