Visiscope csu x1 confocal system

Retinal flatmounts were prepared and the blood vessels were stained using fluorescein-conjugated isolectin B4 (IB4) (Table S1). Immunostaining with IB4 and DAPI was performed for two days at 4 °C. Following incubation with the antibodies, retinae were washed 3 times for 5 min each in phosphate buffer solution (PBS) and were embedded using aqua-poly/mount (18606-20; Polysciences Europe). For analysis, multiplane z-series were collected from fields of views of the vascular network from wildtype and KO mice by using 25× objective lens (VisiScope CSU-X1 confocal system; Visitron Systems). The series included the vitreal surface to the outer plexiform layer (OPL), and we captured at least 4 fluorescent images/retina/central and peripheral areas. Next, we performed a z-stack projection, dividing the z-series in two sets: the superficial vascular plexus (SVP) and the deep vascular plexus (DVP). The retinal vessel density was quantified using the angiotool software [32 (link)].

To analyze the lesion area, the RPE-choroid-sclera complex was stained using two different markers to delimitate the laser injury site—the endothelial cell marker CD31 and the RPE marker β-catenin (evaluation of RPE cell disruption). Images were obtained using a 10X objective lens focusing on the laser spot by confocal microscopy (VisiScope CSU-X1 confocal system, Visitron System). ImageJ (NIH) was used to measure the lesion size in a masked fashion. Lesions were excluded when there was obvious damage due to tissue processing. The mean lesion size obtained from all 3 lesions per eye was plotted as one experimental value per animal and was subjected to further statistical analysis.

Freshly isolated retinal slices (1 mm thickness) were prepared and placed with the photoreceptor side onto membrane filters (0.45 μm, diameter 50 mm; GE Healthcare Life Sciences, Freiburg, Germany). Volume changes of Müller cell somata evoked under isotonic conditions and after hypoosmotic challenge (60% of control osmolarity) were measured in the inner nuclear layer of retinal slices as previously described [77 (link)]. The retinal slices from wildtype and KO mice were placed in a perfusion chamber (custom made) and loaded with the vital dye Mitotracker Orange (10 μM, excitation 543 nm, emission 560 nm-long pass filter; Thermo Fisher Scientific). The Mitotracker Orange dye selectively stained Müller glia [78 (link)], and the stock solution was prepared in DMSO and diluted 1:1000 in PBS. The slices were examined during exposure to a hypotonic solution for 4 min with or without test agents using confocal microscopy (custom-made VisiScope CSU-X1 confocal system equipped with a high-resolution sCMOS camera; Visitron Systems). The cell soma area of labeled Müller cells was cross-sectionally measured (ImageJ). The blocking agent was pre-incubated for 10 min in extracellular solution; AC710 (PDGFR family inhibitor; Tocris Bioscience, Bristol, UK) 100 nM. PDGF-BB, a PDGF receptor α/β agonist (R&D Systems), was applied simultaneously with the hypotonic solution.