Donkey anti goat cy 3

The following primary antibodies were used to stain macrophages/Kupffer cells: rat anti-mouse CD68 antibody (AbDSerotec), rat anti-mouse F4/80 antibody (AbDSerotec), and rat anti-mouse CD169 antibody (AbDSerotec). To stain endothelial cells, rat anti-mouse CD31 antibody (BD Biosciences) was applied. Goat anti-mouse VEGFR2 antibody (R and D Systems) was used to determine the VEGFR2 density. Secondary IgG antibodies (donkey anti-rat Alexa Fluor 488, donkey anti-rat Cy-3 and donkey anti-goat Cy-3) were obtained from Dianova. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Merck KGaA).

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.