Visualizing Retinal Vasculature via Immunofluorescence

Immunofluorescence staining was performed on retinal sections or wholemount preparations in order to visualize the retinal vasculature. Staining was performed following standard protocols as previously described (Fernández-Sánchez et al., 2015a (link)). Briefly, to improve the tissue permeability to the antibodies, wholemount retinas were incubated in 0.02% sodium borohydride (#163314; Panreac, Barcelona, Spain) in PB (5 min, RT). Sections and wholemount retinas were incubated with goat anti-collagen type IV antibodies (1:100; Millipore Cat # AB769) at 4°C temperature overnight or for 3 days, respectively. After washing three times for 10 min in PB, the retinas were incubated for 1 h or overnight, respectively, in Alexa Fluor 488 (green)-conjugated anti-goat IgG from Molecular Probes (Eugene, OR, USA) at a dilution rate of 1:100. Finally, the preparations were washed in PB, mounted in Citifluor (Citifluor Ltd., UK) and coverslipped for viewing under fluorescence microscopy, using a Leica CTR MIC microscope (Leica Microsystems, Germany).