Donkey anti mouse alexa 546

To analyse the ability of transduced and non-transduced HDFneo to generate elastic fibres, cells were seeded into chambered coverglasses (LabTek®, Nunc, Penfield, USA). Cells were grown for 14 days with 10 ng/mL TGF-β1 or left untreated as a control. Afterwards, cells were washed twice with PBS, fixed with methanol:acetone (1:1) at −20 °C and washed again twice with PBS at room temperature (RT). For immunostaining, samples were first incubated with PBS containing 1% BSA (blocking buffer) for at least 1 h at RT and subsequently labelled with the primary antibodies polyclonal rabbit anti human TE/elastin (1:200; Abcam) and monoclonal mouse anti fibrillin-1 clone 11C1.3 (1:100; Dianova GmbH, Hamburg, Germany) in blocking buffer. After three washing steps with PBS at RT, incubation with secondary antibodies was done with donkey anti rabbit Alexa 647 and donkey anti mouse Alexa 546 (both 1:1000; Life Technologies, Carlsbad, USA) in blocking buffer. Counterstaining of cell nuclei was carried out using DAPI (1 μg/mL in PBS; Roche, Basel, Switzerland).

For cryosections, two different pieces of each retina were excised (Figure 1B). One from the superior ora serrata to the optic nerve head and one from the inferior ora serrata to the optic nerve head. These parts were sectioned vertically at 16 µm on a cryostat embedded in OCT medium (Jung, Nussloch, Germany). Immunocytochemical labeling was done as described previously [37] (link).
For flatmount staining, the nasal part of the retina (Figure 1B) was excised. Retinae were washed several times in PBS at RT and then incubated with a mixture of opsin antibodies diluted in 3% normal donkey serum, 1% bovine serum albumin, and 1% Triton X-100 in PBS for three days at 4°C. After several washing steps in PBS the retinae were incubated with secondary antibodies for 2 h at RT in the same incubation solution as before. Thereafter, retinae were washed in PBS, mounted in Aqua Polymount medium (Polysciences) and stored at 4°C for microscopy. For detailed information about primary antibody see Table 1.
The following secondary antibodies were used: donkey anti-rabbit Alexa 488, donkey anti-mouse Alexa 546 (Life Technology, Darmstadt, Germany), donkey anti-mouse Cy3 and donkey anti-goat Cy3 (Dianova, Hamburg, Germany). All were diluted 1∶500.