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D.C. Chung, N.W. Keiser, J.L. Bennicelli, W. Tang, Z. Wei, J. Bennett; Co–Localization of EFEMP1 and TIMP3 in the Developing and Mature Mouse Retina . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1637.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To evaluate the immunofluorescent localization patterns of EFEMP1 and TIMP3 in the developing and mature mouse retina. Methods: Eyes of CD1 mice at developmental stages E14, P2, P7, P12, P14, and 4 months were collected after euthanasia. Eyes were fixed in 4% paraformaldehyde, cryoprotected and sectioned at 10µm. Sections were blocked, incubated with a polyclonal rabbit anti–EFEMP1 antibody generated in our lab and with a mouse anti–human TIMP–3 antibody (Calbiochem, San Diego, CA). After washing, sections were incubated in secondary antibodies anti–rabbit IgG Alexa 488 and anti–mouse IgG Alexa 594 (Molecular Probes, Eugene, OR). The sections were washed and stained with DAPI and fluorescence was evaluated on a Leica fluorescent microscope. Results: EFEMP1 and TIMP3 co–localized in RPE at E14, although EFEMP1 was in the apical portion of the cells and TIMP3 was basal and in Bruch’s membrane. Co–localization was also found in the nerve fiber layer. At P2 and P7, there was increased co–localization in the nerve fiber layer, retinal ganglion cells, and the choroid, but not RPE. At P12 and older, co–localization persisted in the retinal ganglion cell layer. EFEMP1, however, then appeared in the basal portion of RPE and the inner segments of the photoreceptors. Conclusions: EFEMP1 and TIMP–3 co–localize to the inner retina, and depending on development stage, in the RPE. The interaction of these two proteins may play a role in angiogenesis in the retina.
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