Alexa 488

Following fixation, retinal pieces were washed six times in 0.1 M phosphate buffer saline (PBS, pH 7.4) then blocked overnight in phosphate-buffered saline (PBS) containing 0.3% Triton-X 100 (TX-PBS) and 3% normal donkey serum (NDS) at 4°C. After the block, tissues were incubated for 5–7 days in primary antibodies (listed in Table 1) diluted in TX-PBS and 1% NDS at 4°C. Retinas were then washed 6 × 10 min followed by incubation overnight in species-specific donkey-raised secondary antibodies diluted in TX-PBS and 1% NDS at 4°C. Secondary antibodies were conjugated to the fluorescent markers Alexa-488, Cy3, and/or Alexa-647, dilution 1:200 (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Neurobiotin injected cells were visualized with streptavidin conjugated to Alexa-488 or Cy3, dilution 1:200 (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Following final washes, retinas were mounted in Vectashield (Vector Laboratories) on glass slides and cover-slipped for examination.

Brains from newborn mice were fixed for 8 h in Immunofix and clarified with the X-Clarity system (Logos Biosystem, South Korea). Briefly, whole brains were polymerized with acrylamide “hydrogel solution” for 3 h at 37 °C with a vacuum followed by the passive clarification for 48 h at 37 °C in “clearing solution”.
Brains were then subjected to deep-tissue TH immunolabeling by staining with TH antibody (dilution 1:100, Merck Millipore) in PBS/0.3% Triton for 3 days, 1 day of washing followed by incubation with anti-rabbit Alexa 488 for 3 days (Jackson Immunoresearch).
Finally, brains were observed by a two-photon upright LSM 880 Zeiss microscope equipped by W Plan-Apochromat 20x/1.0 DIC (UV) VIS-IR M27 75 mm objective. The fluorochrome was excited with the IR laser set at 860 nm. Z-stack were set with the height of the sections scanning = ~1 μm. Images were then reconstructed using Zen lite 2.3 (Carl Zeiss).

Human foetal retinal tissue was fixed and IF performed on cryostat sections as previously described^{2 (link)}. Sections were reacted against the following primary antibodies: CRX (1:200, Abnova, H00001406-MO2, Lot KB191-4G11), Ki67 (1:200, Abcam, ab15580, Lot GR3375617-1), Recoverin (1:1000, Millipore-Merck, ab5585, Lot 3099956), RXRγ (1:200, Santa Cruz Biotechnology, sc-555, Lot D1013), RXRγ (1:100, Santa Cruz Biotechnology,sc-365252, Lot G1122), SNCG (1:500, Antibodies.com, A121664, Lot 25309) and VSX2 (1:100, Santa Cruz, sc-365519, Lot 000031213). Secondary antibodies were conjugated to Alexa488 (Jackson Immuno Research Laboratories, 115-545-146, Lot 157251 and Thermo Fisher, 21202, Lot 2266877), Alexa546 (Thermo Fisher, 10040, Lot 1622582), Cy3 (Jackson Immuno Research Laboratories, 115-165-003, Lot 86185) and Alexa 647 (Thermo Fisher, 21447, Lot 2175459). All secondary antibodies were used 1:1000 diluted in PBS. Antibody specificity was assessed by omitting the primary antibodies. Images were obtained using a Zeiss Axio Imager.Z1 microscope with ApoTome.2 accessory equipment and AxioVision or Zen software. Between 5 and 10 images were collected from each IF analyses. Images are displayed as a maximum projection and adjusted for brightness and contrast in Adobe Photoshop CS6 (Adobe Systems).