Subsequently, we performed co-labeling experiments for Sox9 and markers corresponding to all other retinal cell types

Subsequently, we performed co-labeling experiments for Sox9 and markers corresponding to all other retinal cell types. entire RPC population. Retinal neuronal specification appears to occur normally. However, postnatal MG cells are lost, thereby implicating in MG cell genesis. MATERIALS AND METHODS Mouse strains (Akiyama et al., 2005) and (Furuta et al., 2000) mice were maintained on a C57BL/6J, 129/SvEv mixed genetic background. (Rowan and Cepko, 2004) mice were Resorufin sodium salt maintained on an FVB, 129/SvEv, C57BL/6J, and SJL mixed genetic background. Analyses of wildtype mouse tissue was from a C57BL/6J, 129/SvEv mixed genetic background. For all those embryonic time course studies, noon on the day when vaginal plugs were observed was designated E0.5. Furthermore, any analysis of mutant and control tissue samples was performed on littermates. All work reported was approved by Resorufin sodium salt the institutional animal care and use committee at the University of Texas, M. D. Anderson Cancer Center. Polymerase chain reaction (PCR) was used to genotype mice from all breeding experiments. mice were genotyped as described previously (Furuta et al., 2000; Rowan and Cepko, 2004; Akiyama et al., 2005). Tissue processing For embryonic retinae, whole heads were Resorufin sodium salt fixed in 4% paraformaldehyde (PFA) for 45 minutes at 4C. For postnatal retinae, animals were transcardially perfused with 4% PFA and the eyes were carefully removed with rat tooth forceps and then submersion-fixed in 4% PFA for 30 minutes at 4C. After fixation, all tissue was washed in 1 phosphate-buffered saline (PBS, pH 7.3) 3 10 minutes at 4C. Next the samples were cryoprotected by immersing in 15% and then 30% sucrose until the tissue sank to the bottom of the tubes. Subsequently the tissue was immersed in a 1:1 answer of 30% sucrose and OCT medium and left at 4C for a couple of hours. Then the tissue was embedded in OCT on dry ice and stored at ? 80C prior to sectioning. Cryosections were cut at 20 m on a cryostat and mounted on superfrost plus slides (VWR Brand, Westchester, PA). In order to make more direct comparisons between mutant and control retinal architecture, all sections were collected at the level of the optic nerve. Immunofluorescence Immunofluorescence on retinal cryosections was performed as described (Wang et al., 2001; Ohtoshi et al., 2004) with slight modifications. A table of all primary antibodies including dilutions and recommendations can be found in Table 1. All secondary antibodies were Alexa Fluor-conjugated antibodies from Molecular Probes (Eugene, OR) and used at a dilution of 1 1:400. For immunohistochemistry, cryosections were allowed to dry completely. Slides were postfixed in 4% PFA for 10 minutes and then washed in 1 PBS-T (PBS + 0.1% Triton X-100) 3 10 minutes at room temperature. Next the slides were blocked in 2% normal horse or goat serum diluted in 1 PBS for 1 hour at room temperature. Primary antibodies were diluted in the same Mouse monoclonal to FLT4 blocking answer and incubated around the slides overnight at 4C in a humid chamber. Next the slides were washed 4 occasions at room heat in 1 PBS. Labeling with the secondary antibodies was performed using the same blocking answer (2% normal serum in 1 PBS) and slides were incubated for 1 hour at room heat. All double-labeling experiments were performed Resorufin sodium salt by mixing primary antibodies followed by labeling with a mixture of the corresponding secondary antibodies. Slides were finally mounted with Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA) made up of DAPI nuclear stain. Unfavorable controls, following the above protocol but without the primary antibodies, were run, consistently showing no cellular labeling. TABLE 1 Antibodies knockin mouse line (Taranova et al., 2006). The antibody used in our study results in a temporal and spatial staining pattern identical with these above-mentioned results. Based on MALDI-TOF and (LC) mass spectrometry, we decided the Sox2 blocking peptide (Santa Cruz) sequence to be from amino acid 277C293 (data not shown). The Sox9 antibody detects the 60C65 kDa Sox9 protein and no other bands on Western blots of mouse brain lysate (manufacturers technical information). Furthermore, RNA in situ hybridization with a probe that spans nucleotides 972C1508 (Wright et al., 1995) labels the E16.5 retina in an identical pattern to that of the Sox9 antibody (Fig. 1E of this study and data not shown). Additionally, conditional ablation of in the developing retina abrogates Sox9 antibody staining, further supporting antibody specificity (Fig. 4 of this study). Open in a separate windows Fig. 1 Sox9 expression throughout retinal development..