Mutations in lead to complete congenital stationary night blindness (cCSNB). PNA labeling was severely reduced in the OPL in mice. This study confirmed the localization of LRIT3 at the dendritic tips of depolarizing bipolar cells in mouse retina and demonstrated the dependence of TRPM1 localization on the presence of LRIT3. Since tested components of the ON-bipolar cell signaling cascade and PNA revealed disrupted localization an additional function of LRIT3 in cone synapse formation is suggested. These results point to a possibly different regulation of Optovin the mGluR6 signaling cascade between rod and cone ON-bipolar cells. ((mutations with lacking or severely reduced b-wave amplitudes in the scotopic and photopic electroretinogram respectively (Neuille retinas to better understand the function of LRIT3. MATERIALS AND METHODS Ethics statements All animal procedures were performed according with the Council Directive 2010/63EU of the European Parliament and the Council of 22 September 2010 on the protection of animals used for scientific purposes and were approved by the French Minister of Agriculture (authorization A-75-1863 delivered on 09th November 2011). All efforts were made to minimize suffering. Animal Care The generation and characterization of the knock-out mouse has been described elsewhere (Neuille and retinal sections with these two sera. One of them Rabbit Polyclonal to MAP3K8 (phospho-Ser400). produced a specific punctate signal in the outer plexiform layer of the retinal sections that was absent on sections. However strong non-specific signal and noise were also present on the whole and sections (data not shown). In order to determine which of the two peptides resulted in the specific staining we performed immunohistochemistry on retinal sections after preincubation of the serum with either the N- or the C-terminal peptide. The specific signal was absent when the serum was preincubated with the peptide localized at the N-terminus but Optovin noise remained (data not shown). Subsequently affinity purification was performed with this peptide to decrease noise and to obtain a more specific antibody. Preparation of retinal sections for immunohistochemistry Mice were killed by CO2 administration and cervical dislocation. Eyes were removed and prepared following three methods. For method 1 we made two slits in a cross within the cornea and placed the eyeball in ice cold 4% (w/v) paraformaldehyde in 0.12 M phosphate buffer pH 7.2 for 1 hour. After three 10-min-washes with ice cold phosphate buffer saline (PBS) we transferred the eyeball to cold 30% sucrose. Finally the lens was removed and the eyecup was embedded in OCT (Sakura Finetek AJ Alphen aan den Rijn The Netherlands) and frozen in a dry ice-cooled bath of isopentane. For method 2 the Optovin anterior segment and lens were removed and the eyecup was fixed in ice cold 4% (w/v) paraformaldehyde in 0.12 M phosphate buffer pH 7.2 for 20 min. The eyecup was washed three times in ice-cold PBS and cryoprotected Optovin with increasing concentrations of ice cold sucrose in 0.12 M phosphate buffer pH 7.2 (10% 20 for 1 h each and 30% overnight). Finally the eyecup was embedded in 7.5% gelatin-10% sucrose and frozen in a dry ice-cooled bath of isopentane. For method 3 we made a hole just behind the and placed the eyeball in 4% (w/v) paraformaldehyde in 0.12 M phosphate buffer pH 7.2 for 5 min. We then removed the lens and the eyecup was again fixed for 20 min in paraformaldehyde at room temperature. The eyecup was washed three times in PBS and cryoprotected with increasing concentrations of ice cold sucrose in 0.12 M phosphate buffer pH 7.2 (10% for 1 Optovin h and 30% overnight). Finally the eyecup was embedded in 7.5% gelatin-10% sucrose and frozen in a dry ice-cooled isopentane bath. Sections were cut at a thickness of 18 μm on a cryostat and mounted onto glass slides (Super-Frost Thermo Fisher Scientific Waltham MA USA). The slides were air dried and stored at ?80°C. Immunostaining of retinal cryosections Primary antibodies used for immunostaining are listed Optovin in Table 1. The TRPM1 immunoreactive serum from a patient suffering from melanoma-associated retinopathy (MAR) the rat mGluR6 antiserum raised in sheep the purified goat polyclonal antibody to mouse Gβ5 and the mouse RGS11 antibody raised in rabbit were used as previously described (Chen retinal sections was adapted from a previously published protocol (Ramakrishnan and.