Díaz NM , Morera LP , Verra DM , Contin MA , Guido ME .

	Figure 1. Immunocytochemistry for immunopurified RGC primary cultures at embryonic day 8 (E8) by Thy-1 (a), Opn4x (b) antibody purifications, and clock genes of RGC Opn4x cultures. (a) Primary cultures of embryonic RGCs purified by Thy-1 antibody immunopanning and maintained for 48–72 h were immunolabeled for DM1A, Opn4x, Thy-1, Rhod-4D2, and NeuN. (b) Immunocytochemistry for Opn4x- Prox-1, GABA, glutamine synthetase (GS), and NeuN, with DAPI staining in purified RGC cultures at E8 by Opn4x- antibody immunopurification. Primary cultures were visualized by confocal microscopy with specific primary antibodies as described in Section 2. Scale bar = 20 μm. (c) Primary cultures of RGC's immunopurified by Opn4x-antibody immunolabeled with anti-Bmal1, Cry1, and Per2.4.
	Figure 2. Melanopsin expression and light responses in RGC cultures. (a) Primary cultures of embryonic RGCs maintained for 48–72 h were immunolabeled for Opn4x or Opn4m with specific antibodies and DAPI (nuclei staining). The white arrows identify clusters of Opn4 (+) cells present in RGC cultures observed at 40X. (b) Content of inositol phosphates (IPs) in primary cultures of RGCs maintained in the dark (black squares) or stimulated by light (gray squares). Cultures previously incubated with myo-3H inositol for 48 h were light-stimulated during 90 sec to investigate the generation of different IP derivatives directly in RGCs. IP production was evaluated after bright light stimuli and control cells were maintained in the dark as described in the text. Significant increases in radiolabeled IP3 and IP were seen in cultures exposed to bright white light as compared with dark controls (P < 0.04). On the contrary, no significant light-dark differences were found in the content of other IPs determined such as IP2, IP4, and IP5/6. See text for further details.
	Figure 3. Immunocytochemistry for Prox-1, Opn4x, and α-Tubulin proteins staining and merge in HC cultures at E15. Individual cells from primary cultures of HCs were obtained by the 2.5% phase of a bovine albumin serum (BSA) gradientpurification, maintained for 48–72 h, immunolabeled for Opn4x-like protein ((b) red), α-Tubulin (DM1A) ((a) green), Prox-1 ((a) red; (b) green), and nuclei staining by DAPI (blue), and then visualized by confocal microscopy as described in Section 2.
	Figure 4. Analysis of mRNA expression in primary cultures of chicken RGCs at E8 (a) and of HCs at E15 (b) and in the whole postnatal chicken retina. (a) Chicken embryonic retinas were dissected out at E8 and RGCs were purified and cultured. Expression of clock genes cryptochrome 1 (Cry 1), Clock, Bmal1, NPAS2, and Per 2 and clock-outputs: the melatonin synthesizing enzyme, arylalkylamine N-acetyltransferase (AA-NAT) mRNA was assessed by the reverse transcription- (RT-) polymerase chain reaction (PCR). Cry 1, Per2 Clock, Bmal1, and AA-NAT PCR products were found in RGC cultures whereas no detectable amplification was found for the NPAS2 transcript. (b) Chicken embryonic retinas were dissected out at E15, and HCs were purified and cultured. mRNA expression for clock genes cryptochromes 1 (Cry 1) and 2 (Cry 2), Bmal 1, and Clock and for the clock-outputs AA-NAT was assessed by RT-PCR from HCs at E15 (phase 2.5%) and samples from the whole postnatal retina (positive control). Positive amplification was found for the mRNAs of Cry 1, Per2, Bmal1, and AANAT whereas Cry2, NPAS2, and Clock amplifications products were not found in HC cultures.

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