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Sharon L Oltjen, Leonard M Hjelmeland, Roxana A Radu; In Situ Localization of Complement Factor H Gene Expression in the Mouse Eye. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6327.
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To identify the cells expressing complement factor H in the mouse eye using a novel in situ hybridization assay.
Eyes from C57BL/6 (National Institute on Aging), Cfh-/- knockout and their background strain 129/Sv (Jules Stein Eye Institute, UCLA) were fixed in freshly prepared 10% neutral buffered formalin, embedded in paraffin and sectioned at 6 microns. In situ hybridization (ISH) was performed using the RNAscope 2.0 FFPE Red Assay (Advanced Cell Diagnostics, Hayward, CA) utilizing the manufacturer’s modifications for eye tissue. The Cfh probe was designed by the manufacturer to not cross react with other Cfh related family members. An RPE65 probe was used as a positive eye specific control, along with the manufacturer’s positive and negative controls. Western blots were performed using either goat α-mouse Factor H or rabbit α-mouse Factor H antibodies (Santa Cruz Biotechnology).
We have determined the location of Cfh mRNA in the mouse eye using a novel in situ hybridization assay. Cfh mRNA was expressed in the retinal pigment epithelium (RPE), inner nuclear layer (INL), ganglion cell layer (GCL), posterior ciliary arteries and occasionally in some other cells in the retina of C57BL/6J and 129/Sv mice. No expression was detected in the Cfh-/- mouse eye. Expression was highest in the RPE>INL>GCL. Pigment and poor choroidal morphology made it difficult to visualize an ISH signal in the choroid. The positive control RPE65 probe only labeled the RPE. Western blot analysis of serum samples from C57BL/6 and 129/Sv showed protein expression with a band for Cfh at ~155 kDa. This band was completely absent in Cfh-/- serum samples.
Cfh mRNA expression in the posterior eye was observed mainly in the RPE, followed by the INL and to a lesser extent the GCL. It was not possible to observe a signal in the choroid due to pigment and poor choroidal morphology.
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