The distance between the strips was chosen such that outgrowing temporal and nasal RGC axons came into contact within 24–36 hr. Strips were cut perpendicular to

the temporonasal DAPT clinical trial axis and thus contained either temporal or nasal RGCs. For the time-lapse analysis, we used a Nikon Eclipse Ti-E inverted microscope and Cool SNAP HQ2 camera. The interaction between temporal and nasal axons was analyzed for 3–10 hr. Pictures were taken with a 10× lens every minute with the entire area between the two strips documented. For this, pictures were taken from overlapping areas and stitched together using NIS software. Routinely, an area of about 4 × 2 mm was recorded. For data analysis, the area between nasal Selleck Lapatinib and temporal strips was subdivided into 10–18 regions of interest using ImageJ, analyzed individually, and then pooled. We only analyzed axons which could be clearly identified as single growing axons for > 30 min before contact with other axons. Only the first contact was counted for each axon. Furthermore, we only included axons in the analysis which clearly advanced prior to contact and which had a clearly visible growth cone. The interactions were scored as

follows: “0”, no growth cone collapse (axon crosses another axon without growth cone collapse and no/very little change in growth speed) (representative Movie S1); “0.3”, a short transient growth cone collapse after contact and/or a clear slowing down of growth speed, but eventual crossing of the other axon (Movie S2); “0.6”, a full growth cone collapse after contact (Movie Non-specific serine/threonine protein kinase S3); “1”, a full growth cone collapse with a strong retraction of the axon (Movie S4). The recordings were analyzed by two individuals independently and blind to the identity of the class of axons analyzed. Immunohistochemical analysis of frozen sections was performed using standard procedures. Nonspecific interactions were blocked with

1% BSA-TBST, primary antibody solution was applied overnight, and secondary antibody solution was applied for 2 hr, all at room temperature. RNA was extracted from littermate pups on the day of birth using standard protocols. For retina, RNA was extracted from the whole retina of one eye. For SC, RNA was extracted from the central third of the SC from one side. RNA was then reverse transcribed and PCR performed to detect the relative abundance of ephrinA5 expression levels (ephrinA5 FW: TTT GAT GGG TAC AGT GCC TGC GAC; ephrinA5 Rev: AAG CAT CGC CAG GAG GAA CAG TAG) or β-actin (β-actin FW: GAT GAC GAT ATC GCT GCG CTG GTC G; β-actin Rev: GCC TGT GGT ACG ACC AGA GGC ATA CAG) using the following protocol: 94°C, 5 min, 30× (94°C, 1 min; 60°C, 1 min; 72°C, 1 min) followed by 72°C, 10 min. Genomic DNA was extracted using the HotSHOT method (Truett et al., 2000), and genotyping reactions were performed for the presence of ephrinA5 wild-type, KO-first, and floxed alleles as well as rx:cre and en-1:cre alleles.