Mab1563

Retinas for whole-mount were fixed in 4% PFA for 2 hours at room temperature, or overnight at 4 °C. After fixation, retinas were blocked and permeabilized in blocking buffer (1% BSA and 0.5% Triton X-100 in PBS) overnight at 4°C. For mouse antibodies, samples were washed and blocked with mouse IgG blocking reagent as suggested by the manufacturer (MKB-2213 Vector, CA, USA) for 1 hour at room temperature. Primary antibodies were diluted in blocking buffer and incubated overnight at 4°C. The following antibodies were used: VE-cadherin (1:200, AF1002 R&D systems, Abingdon, UK), α-smooth muscle actin-cy3 (1:500, C6198 Sigma, Taufkirchen, Germany), N-cadherin (1:200, 33-3900 lifetechnologies, CA, USA), collagen IV (1:500, 1340-01 southern biotech, AL, USA), presenilin 1 (1:200, MAB1563 Millipore, Darmstadt, Germany) and desmin (1:500, ab15200 abcam, Cambridge, UK). For secondary detection Alexa Fluor-coupled secondary antibodies (1:200) were used. Cell nuclei were visualized with DAPI (0.2 ug/mL, D9542 Sigma). After antibody staining retinas were post-fixed with 4% PFA for 10 minutes before flat-mounting in mounting medium (Dako).