Adult fish were overdosed with tricaine methane sulfonate and eyes were enucleated, followed by removal of the lens and then immersion in fresh 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4 for ~16 hrs. After fixation, samples were cryoprotected in phosphate-buffered 20% sucrose before embedding with Tissue-Tek O.C.T. compound (Sakura, Finetek). Embedded samples were kept frozen at −80 °C until sectioned to 8 microns on a CM3050S cryostat (Leica). Sections were collected on Superfrost/Plus slides (Fisher Scientific), dried and stored at −80 °C. Immunohistochemistry was performed as previously described4 (link), 38 (link) using the following primary antibodies: rat anti-BrdU (dividing cell marker, 1:400; Abcam); rabbit anti-GFP (1:1000; Invitrogen); mouse anti-glutamine synthetase (GS) (Müller glia marker, 1:500; Chemicon/Millipore). Secondary antibodies were conjugated to Alexa Fluor 488 and used at the following dilutions: 1:500 for anti-mouse, 1:250 for anti-rat and 1:1000 for anti-rabbit. For BrdU staining, sections were pretreated with 2N HCl for 20 min at 37 °C and then soaked in 100 mM sodium borate for 10 min. Following immunhistochemical staining, slides were rinsed with water and allowed to dry in the dark prior to cover-slipping with 2.5% PVA (PVA-polyvinyl alcohol)/DABCO (1,4 diazabicyclo [2.2.2]octane). Slides were examined in a Zeiss Axiophot fluorescence microscope equipped with a digital camera or an Olympus FluoView FV1000 confocal imaging system.
Combined in situ hybridization and antibody staining were performed on retinal sections as described previously39 (link). Double in situ hybridizations were done according to manufacturer’s instructions (Perkin Elmer). Sense control probes were generated and showed no signal above background (data not shown). In situ hybridization for let-7a miRNA expression was performed using a zebrafish let-7a LNA probe (Exiqon). The probe was diluted to 1 μM in prehybridization buffer and hybridization and wash conditions were as previously described40 (link).
Combined in situ hybridization and antibody staining were performed on retinal sections as described previously39 (link). Double in situ hybridizations were done according to manufacturer’s instructions (Perkin Elmer). Sense control probes were generated and showed no signal above background (data not shown). In situ hybridization for let-7a miRNA expression was performed using a zebrafish let-7a LNA probe (Exiqon). The probe was diluted to 1 μM in prehybridization buffer and hybridization and wash conditions were as previously described40 (link).
