Figure 3.

Quantitative RNA-seq analysis and immunofluorescence reveal the presence of SPP1 in human CNV tissue. (A) Experimental scheme. RNA was extracted from formalin-fixed and paraffin-embedded human healthy choroid and CNV membranes for massive analysis of cDNA ends (MACE) RNA-seq analysis. Retinal pigment epithelium is abbreviated with RPE. (B) SPP1 expression was quantified by RNA-seq (MACE) in four control samples and four CNV membranes (p = 0.019). Bars represent mean ± s.e.m. (C) Immunofluorescence for DAPI (white), SPP1 (green), and IBA1 (red) in the retina and RPE/choroid of one body donor and in a CNV membrane of a patient diagnosed with neovascular age-related macular degeneration. IBA1⁺SPP1⁺ macrophages (arrow) could be found in both healthy RPE/choroid and CNV membranes in line with the RNA-seq (MACE) results, next to IBA1⁺SPP1⁻ macrophages (asterisk). Arrowheads point to IBA1⁻SPP1⁺ cells. Dashed white squares indicate the region of interest shown as magnifications on the right side. The layering is indicated for the inner and outer plexiform layer (IPL, OPL), inner and outer nuclear layer (INL, ONL), retinal pigment epithelium (RPE), and choroid.