Laser capture was performed as previously described [69]. Total RNA was isolated using a Pico Pure extraction kit (Arcturus) according to manufacturer’s protocol. The optional DNAse step was omitted, and primers that span introns were used for analysis. Two rounds of amplification were used to increase sensitivity while mitigating background amplification. For Rp1, the F1/R1 primer pair was used for both rounds of amplification, whereas for Clrn1, F2/R2 primers were used for the first round, and F1/R1 primers were used for the second round of amplification (see Figure S1). The second round of amplification used 1/100th of the first round PCR product as template for the second round of PCR. Reverse transcription was performed as described above. Retinal Histology Eyes for histological analysis were processed as previously described [70]. Eyes were isolated from 9 month old littermate Clrn1 KO and age-matched WT C57BL/6J animals after cardiac perfusion with 2.5% formaldehyde/2% glutaraldehyde in PBS (pH 7.4). Eyes were immersion fixed for at least 24 hours. The samples were dissected, osmicated, washed, and dehydrated in a graded ethanol and embedded in Epon-Araldite resin (Ted Pella, Inc., Redding, CA). Following polymerization, 1 mm sections were cut on a microtome (UltraCut; Leica, Deerfield, IL) and stained with 1% toluidine blue. Images were digitally captured with a CCD camera (Axiocam; Carl Zeiss Meditec, Inc., Thornwood, NY) on an Axiophot II photomicroscope (Carl Zeiss, Oberkochen, Germany). Clarin 1 Antibody Production Several antibody production technologies were used to generate reagents for immunological analyses. We generated two rabbit antibodies (Sigma-Genosys/Sigma-Aldrich, St. Louis, MO) to two human CLRN1 peptides (59-ASGQELDKFMGEM(C)-39; aa (amino acids) 49–61; ‘‘CLRN1-A’’) and (59-(C)GFQFPFAKSK- DAE-39; aa 209–222; ‘‘CLRN1-B’’). New Zealand White rabbits were injected with peptides conjugated to keyhole limpet hemocyanin (KLH). Serum from the B-peptide showed high specificity in vitro, and CLRN1-B2 antibodies were affinity purified and used in immunohistochemistry, western blotting, and IP experiments. We also generated chicken IgY antibodies by immunizing chicken eggs with two mouse peptides (59- KVHRLSEKIANFKE-39; aa 160-173; ‘‘Clarin160’’) and (59- PFTKSKETETTNVA-39; aa 214-227; ‘‘Clarin214’’). Both anti- bodies were affinity purified and tested in transfected HEK293 cells, western blotting, and immunohistochemistry. Immunocytochemistry Standard immunocytochemical procedures were followed. Procedures were performed at room temperature (RT) unless indicated otherwise. Briefly, after fixation, the cells were rinsed once in phosphate buffered saline (PBS, pH 7.4) and twice with PBT (PBS including 0.1% Triton X-100 detergent (Sigma-Aldrich) and 0.1% bovine serum albumin (BSA; Sigma-Aldrich)). Antibody dilution buffer (ADB) was made by adding 2% BSA and 1:100 normal donkey serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) to PBT. The cells were blocked for 30 minutes in ADB, then primary antibodies diluted in ADB were added following removal of the blocking buffer. After one hour, the antibodies were removed and the cells were rinsed with PBT 36109. Finally, Cy3 conjugated secondary antibodies (Jackson ImmunoResearch) diluted 1:500 in ADB were added to the cells for 309. After rinsing again 36109 with PBT and 2659 with PBS, the cover glass with cells attached was inverted onto a clean microscope slide containing a drop of Vectashield hard set Clarin 1 Is Necessary for Hearing but Not Vision mounting medium including DAPI (Vector Laboratories, Inc., Burlingame, CA). Cells were photographed on an Axiophot II photomicroscope (Carl Zeiss) fitted with an AxioCam digital camera and fluorescence filters for three color emission. Images were identically post-processed using Adobe Photoshop (Adobe Systems). Cryosection Tissue Processing and Immunohistochemistry WT and Clrn1 KO mouse eyes were isolated following euthanasia by CO2 overdose. Eyes were removed rapidly and fixed in 10% neutral buffered formalin (NBF; Ted Pella, Redding, CA) for at least 24 hours, rotating at 4uC. Eyes were opened and the cornea, lens, and vitreous were removed, leaving the posterior pole containing the retina. The posterior pole was rinsed in PBS and equilibrated into 30% sucrose (in PBS) overnight. Eyecups were embedded in Tissue Freezing Medium (TFS; TBS, Durham, NC), and 12–16 mm thick sections were mounted on probe-on plus microscope slides and air-dried. For histology, the tissue sections were counterstained with hematoxylin. For cryosection immunohistochemical procedures, we utilized essentially the same reagents and concentrations as for immunocytochemistry, except that incubation times were increased due to the diffusion of reagents into and out of the thicker tissue samples. For CLRN1-B2 immunohistochemistry (Figure S5), 12 mm thick cryosections were used. Briefly, sections were thawed, blocked with 5% normal donkey serum in PBT and incubated with primary antibodies diluted in PBT overnight at 4uC. Sections were rinsed and incubated in secondary antibodies (anti-rabbit Cy3), rinsed again and visualized on a Zeiss Axiophot 2 confocal microscope (Carl Zeiss). Thick-Section Confocal Microscopy Tissue processing for confocal microscopy was performed as described previously [71]. Briefly, eyes and subsequently anterior eye structures were removed, and posterior poles were immersion fixed in 10% NBF for at least 2 hours. Whole retinas were gently removed from the eyecups following severing of the optic nerve head. Retinas were rinsed well with PBS, and embedded in low- melting temperature agarose (5%) at ,40uC. After cooling, the agarose blocks were sectioned on a Leica vibratome at 100 mm, and collected in PBS. Sections were selected and placed into antibody dilution buffer (ADBX (TNTB; 100 mM Tris (pH 7.4), 150 mM NaCl, 0.1% BSA, 0.1% Triton X-100) plus 1% BSA and 1% normal donkey serum). Sections were incubated for at least 16 hours at 4uC with gentle rocking. The following day, the blocking solution was removed and primary antibodies diluted in ADBX were added for another ,16–24 hour incubation, rocking at 4uC. Several primary antibodies were used, including: the lectin peanut agglutinin (PNA)-Rhodamine (Sigma-Aldrich, St. Louis, MO), anti-synaptophysin (mouse monoclonal, 1:500, DAKO North America Inc., Carpinteria, CA), anti-vimentin (mouse monoclonal, 1:500, DAKO), anti-GFAP (rabbit polyclonal, 1:500, DAKO), anti-PKCalpha (rabbit polyclonal, 1:250, Biomol, Ply- mouth Meeting, PA), anti-Calbindin (rabbit polyclonal ab11426, 1:250, Abcam Inc., Cambridge, MA), and custom anti-CLRN1 antibodies (see above) generated in rabbits and chickens (1:100– 1:500). The following morning, the primary antibodies were removed, and the tissue sections were rinsed briefly with PBS, 36159 in TNTB, and once for 2 hours in TNTB (rocking during each rinse). Following the rinsing, sections were incubated for ,16 hours in Cy3 conjugated secondary antibodies diluted in ADBX. Sections were again rinsed as above, followed by an additional ddH2O rinse. Sections were then counterstained with PLoS Genetics | www.plosgenetics.org 14 August 2009 | Volume 5 | Issue 8 | e1000607