Cy2 affinipure donkey anti rabbit

Eyeballs were dissected and fixed in 4% paraformaldehyde (Cat. No. BM-698; Bostonbioproducts, BioProducts, Ashland, MA) for 2 hours at room temperature followed by immersing into 20% sucrose solution in phosphate buffered saline (PBS) for 2 hours. The eyeballs were embedded in O.C.T. compound (Cat. No. 4583; Sakura Finetek USA, Inc., Torrance, CA) on dry ice. Frozen sections of the retina (10 μm) were incubated with a blocking buffer containing 4% bovine serum albumin and 0.5% Triton X-100 in PBS for 1 hour followed by incubation with a primary antibody against β-III-tubulin (1:500; Cat. No. MAB5564, Millipore), Recoverin (1:500; Cat. No. AB5585, Millipore) or H3K27me3 (1:500; Cat. No. 9733S, Cell Signaling Technology, Danvers, MA) in the blocking buffer for overnight at 4°C. Slides were washed with PBS 3x at 10 min. each before incubation with Cy3/Cy2-conjugated secondary antibody in the blocking buffer [Cy3-AffiniPure Donkey Anti-Mouse (1:500; Cat. No. 715-165-151, Jackson ImmunoResearch) or Cy2-AffiniPure Donkey Anti-Rabbit (1:500; Cat. No. 711-095-152, Jackson ImmunoResearch)] were applied for 1 hour at room temperature. Slides were washed with PBS 3 x at 10 min. each. The slides were mounted in a mounting media containing DAPI (Cat. No. H-1200; Vector Laboratories Inc.) and imaged with a TSC SP5 confocal microscope (Leica Microsystems, Richmond, IL).