Sections were incubated in blocking solution (1% BSA/0.1 Triton-X100 in PBS) for 1 hour at RT and then in primary antibody in blocking solution overnight at 4°C. Slides were rinsed in blocking solution 3 times and incubated with secondary antibody in blocking solution for 2 hours at RT. Slides were rinsed with PBS, mounted in Fluoromount-G and visualized on a Leica SP5 X MP confocal microscope.
Primary antibodies used were anti-Calbindin28K (1∶2000) (Swant, Switzerland), anti-Chx10 (1∶1000) [30] (link), anti-Glutamine Synthetase (1∶10,000) (Sigma), anti-Rhodopsin (1∶100) [31] (link), anti-GFAP (1∶100) (Abcam), and anti-HNF-6/OC1 (1∶200) (Santa Cruz Biotechnology). The anti-Pax6 (1∶50) and anti-Islet1 (1∶50) antibodies were obtained from the Developmental Studies Hybridoma Bank (DHSB), developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biology, Iowa City.
Primary antibodies used were anti-Calbindin28K (1∶2000) (Swant, Switzerland), anti-Chx10 (1∶1000) [30] (link), anti-Glutamine Synthetase (1∶10,000) (Sigma), anti-Rhodopsin (1∶100) [31] (link), anti-GFAP (1∶100) (Abcam), and anti-HNF-6/OC1 (1∶200) (Santa Cruz Biotechnology). The anti-Pax6 (1∶50) and anti-Islet1 (1∶50) antibodies were obtained from the Developmental Studies Hybridoma Bank (DHSB), developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biology, Iowa City.
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