After 5 min incubation time with Evans Blue dye, fluorescence is largely limited to the NFL (a). Performing PCR analysis, we showed expression of claudin-1, 3, 5a, 5b, 9, 11, and 19 in the fish retina, claudins that typically occur at brain barriers or myelin. We could show by immunostains for doublecortin, a marker for differentiating neurons, that new axons are not surrounded by the myelin-like wrappings but only by the endfeet of MCs. We hypothesize that the Saridegib tight junctions in Saridegib the NFL of fish might contribute to the separation of an extracellular space around axons facilitating conductance, from a growth-promoting environment. For a functional test we applied Evans Blue dye to eye cup preparations which showed a retention of the dye in the NFL. This indicates that these remarkable tight junctions can indeed act as a diffusion barrier. to elucidate structural components of the interface between the neural retina including the NFL and vitreous. We hypothesized that cellular structures directly surrounding growing axons differ Saridegib from the surroundings of existing axons. The retina of has been extensively studied previously, in addition to behavioral, social, and genetic aspects of this species (Mack and Fernald, 1995; Renn et al., 2008; Fernald, 2012; Mack and Tiedemann, 2013). This species has the advantage of rapid and extensive growth over several years. We first used several histological approaches including ultrathin section and freeze-fracture electron microscopy, and subsequently molecular methods to reveal unusual tight junctions in the NFL. Materials and Methods Animals For this study, we used eyes of the cichlid fish bred in our own colony. All procedures were performed according to governmental guidelines and were approved by local authorities (Regierungspr?sidium Tbingen). We used animals of either sex between 3 months and 1 year of age with a standard length of 3C5 cm. For the removal of the eyes, animals were anesthetized with MS222 and killed by cervical section. Immunohistology For immunohistological stains, excised eyes were fixed in 4% paraformaldehyde after removal and processed as previously described (Mack et al., 2004). Briefly, eyes freed of the lens and sclera were rinsed and cryoprotected in 30% sucrose before frozen and sectioned on a cryostat at 18 m. Primary antibodies were applied on sections overnight at 4C after preincubation in normal goat serum. See Table ?Table11 for the detailed information on antibodies used. After three washes in PBS, secondary antibodies were applied for 1.5 h, rinsed again and coverslipped in Mowiol. In some stains we applied the nuclear stains Sytox Green (excited at 488 nm) or DRAQ5 (excited at 633 nm; both from Thermo WAF1 Fisher Scientific). Sections were examined on a LSM510 confocal microscope (Zeiss, Oberkochen, Jena, Germany), using laser excitations at 488, 543, and 633 nm in sequential scans with appropriate filter sets. The images were captured with ZEN 2009 software (Zeiss), linear contrast and brightness adjustments and assembling of image plates were carried out with Adobe Photoshop CS4. Table 1 List of antibodies used. for 30 s. The upper, nucleic acid-containing, aqueous phase was pipetted in a fresh tube and total RNA isolation was automated in the QIAcube (Qiagen) using the RNeasy Plus Universal Mini Kit (Qiagen) following manufacturers protocols. The RNA concentration of all samples was measured with the Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Darmstadt, Germany). RNA was converted into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen) following manufacturers description. PCR was performed with 2 l samples of the RT-reaction, 0.2 M of the forward and reverse primers, 0.2 mM dNTP, 2 mM MgCl2 and 1.25 U Taq polymerase (VWR Life Technology Competence Center). All primers (observe Table ?Table22) were specifically designed for predicted mRNA-sequences, which were retrieved from National Center for Biotechnology Info (NCBI), using DNASTAR Software. PCR was performed for 35 cycles: 30 s denaturation at 95C, followed by 40 s annealing at 62C, and 1 min elongation at 72C. The PCR products were analyzed with standard electrophoresis on 1.5% agarose gels at 100 V, stained with peqGREEN (VWR Life Technology Competence Center) and photographed under UV illumination having a gel documentation system (E-Box VX2, Vilber Lourmat, Eberhardzell, Germany). The size of each PCR product was estimated by using a peqGold 100 bp DNA Ladder (VWR Existence Science Competence Center). Table 2 Primers utilized for RT-PCR analysis. claudin gene sequences with likely expression, specifically for claudin-1, claudin-3, claudin-5a, claudin-5b, claudin-9,.