May 1994
Volume 35, Issue 6
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Articles  |   May 1994
Immunolocalization of the retinoblastoma protein in the human eye and in retinoblastoma.
Author Affiliations
  • T M Nork
    Department of Ophthalmology, University of Wisconsin Medical School, Madison 53792-3220.
  • L L Millecchia
    Department of Ophthalmology, University of Wisconsin Medical School, Madison 53792-3220.
  • G Poulsen
    Department of Ophthalmology, University of Wisconsin Medical School, Madison 53792-3220.
Investigative Ophthalmology & Visual Science May 1994, Vol.35, 2682-2692. doi:
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      T M Nork, L L Millecchia, G Poulsen; Immunolocalization of the retinoblastoma protein in the human eye and in retinoblastoma.. Invest. Ophthalmol. Vis. Sci. 1994;35(6):2682-2692.

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Abstract

PURPOSE: To determine whether, by employing recent advances in immunocytochemical technique, it is possible to identify reliably the product of the retinoblastoma (RB) susceptibility gene, p110RB1, in formalin-fixed, paraffin-embedded eyes with commercially available primary antibodies. If so, the authors sought to determine the distribution of p110RB1 in normal human eyes and retinoblastomas in hopes of better understanding its function. METHODS: Four antibodies to p110RB1 were tested on normal human and monkey eyes, as well as on six human retinoblastomas. The human tissue was formalin-fixed and paraffin-embedded. Free antigen was used for an absorbed control. The monkey eye had been injected with tritiated (H3) thymidine 24 hours before enucleation. RESULTS: Three of the four antibodies had acceptable reactivity (a polyclonal against the carboxyl-terminal epitope and two monoclonals against epitopes near the amino-terminus). Staining was confined to nucleated cells of the normal eyes and was strongest in the cycling cells of the lenticular and corneal epithelia. Somewhat weaker reactivity was seen in those corneal epithelial cells in S phase as determined by autoradiography for H3-thymidine. Of the six retinoblastomas, three had strong nuclear and cytoplasmic staining and one showed weaker staining in the tumor cells than in the adjacent vascular endothelial cells. Two of the tumors had positive cytoplasmic and negative nuclear staining with an amino-terminal antibody but were completely negative for carboxyl-terminal p110RB1 reactivity. CONCLUSIONS: Using appropriate immunocytochemical techniques, p110RB1 can be identified in paraffin-embedded tissues with commercially available antibodies. The observed staining pattern in retinoblastoma suggests that RB1 transcripts are commonly produced in the tumor cells and that they are sometimes, but not always, capable of nuclear binding. Thus, nuclear binding by the RB1 gene product per se is not sufficient to prevent tumor growth, nor does it indicate the presence of a normal transcript.

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