Immunohistochemistry All http://www.selleckchem.com/products/Abiraterone.html procedures used were reported previously (46, 47). Cross-sections of mouse eyecups were incubated with primary antibodies, namely anti-rabbit red/green pigment opsin, anti-rabbit opsin blue anti-mouse rhodopsin, anti-mouse PS, anti-rabbit annexin V, and PNA. Signals were detected with either Cy3-conjugated secondary antibody or Alexa488-conjugated secondary antibody. Nuclear staining was achieved with DAPI. Sections were analyzed with a Leica 6000B microscope. Whole-mount retinal confocal microscopy Ten Wt and 10 Nrl-deficient 4-wk-old mice were sacrificed, and eye whole mounts were prepared and incubated overnight with primary antibodies; i.e., anti-rabbit red/green pigment opsin, anti-rabbit opsin blue anti-mouse rhodopsin, anti-mouse PS, and PNA.

Signals were detected with either Cy3-conjugated secondary antibody or Alexa488-conjugated secondary antibody. Nuclear staining was achieved with ToPro3. Thick Z stacks (30�C40 ��m) were collected at ��40 view with 1 ��m between each slice and visualized with a Leica SP5 confocal microscope. Obtained images were postprocessed with ImageJ to adjust contrast and brightness. Scanning EM (SEM) Seven Wt and 9 Nrl-deficient mice 4 wk of age were sacrificed, and their retinas and the RPE were separated and fixed in 2.5% glutaraldehyde, 0.1 M cacodylate buffer, and 2% sucrose (pH 7.4) for 24 h. Samples were washed in 0.1 M cacodylate buffer and 2% sucrose, fixed with 1% OsO4 in washing buffer, dehydrated with ethanol, dried by a critical point drying method (48), and sputter-coated with a 5�C10 nm gold layer.

Samples were imaged with a JSF-6300F scanning electron microscope (Jeol, Akishima, Japan) at the University of Washington Department of Pathology (Seattle, WA, USA). The emission current was set to enable acquisition of backscattered electron scanning images at ��2000 to ��10,000. Transmission EM (TEM) Five Wt and 5 Nrl-deficient mice aged 4 wk and 5 Wt and 5 Nrl-deficient mice aged 8 wk were sacrificed at 1.5 h after lights went on in the morning. Eyes were removed, and whole eye cups were dissected out under a surgical microscope and placed in 4% paraformaldehyde at 37��C for 4 h. Eye cups then were rinsed in PBS and incubated in a 1:1 solution of 2% OsO4:3% potassium ferrocyanide for 1 h. This was followed by incubation in a new mixture of 2% OsO4:3% potassium ferrocyanide for 1 h, after which eye cups were washed in filtered water and placed in 0.

25% uranyl acetate overnight at 4��C. Eye cups were dehydrated the next day for 10 min each in sequential solutions of 30, 50, 75, 85, 95, and 100% ethanol in water, then for 15 min each in sequential solutions of 50, 75, and 100% propylene oxide in ethanol, followed by 2 h in 30% epon in propylene oxide, and Dacomitinib finally kept in 50% epon in propylene oxide overnight.