Reporter visualization. Mice were sacrificed by CO2 asphyxiation, and their eyes were isolated and typically fixed in 2% paraformaldehyde in sodium acetate buffer, pH 6.0 [12 (link)]. The corneas surgically dissected from the P0-Cre;R26-tdTomato animals were fixed in 4% paraformaldehyde and examined directly following mounting, without antibody-mediated enhancement. In the case of the MADM mice, whole corneas were incubated overnight at 4 °C in combined primary antibodies (chicken anti-green fluorescent protein [GFP]; Aves Labs, Tigard, OR; 1:500, and goat anti-c-Myc; Novus Biologicals, Littleton, CO; 1:200) diluted in Tris-buffered saline (TBS), pH 7.3, with 1.0% bovine serum albumin (BSA) and 0.4% Triton X-100. Anti-c-Myc antibody was preabsorbed with fixed wild-type tissue before use [13 (link)]. The following day, the slides were labeled serially with fluorescein isothiocyanate (FITC)–conjugated and Alexa Fluor 555–conjugated secondary antibodies (1:200 and 1:400 dilution, respectively) to enhance visualization of GFP and c-Myc-RFP, respectively. After several buffer rinses, four radial incisions were made in each cornea to produce “petals,” and the resulting flatmounts were placed on glass slides with the endothelium facing up and then coverslipped using Vectashield fluorescence mounting medium (Vector Labs, Burlingame, CA) [14 (link)].