Five-micrometer sections of all globes were also prepared for immunofluorescence staining by fixation in formalin and 1X phosphate buffered saline (PBS; 127 mM NaCl, 2.7 mM KCl, and 10 mM phosphate) at 25°C. After fixation, globes were embedded in paraffin, sectioned, and mounted on glass slides. Sections were examined 160 μm to the left and right of the peak cell count as previously determined through histologic cell counts. Two sections per eye of the Brn3b -/-, Brn3b +/-, and Brn3b +/+ mice were stained. Primary antibodies were: rabbit anti-calretinin (1:100 dilution, #232A-74; Cell Marque, Rocklin, CA) and mouse anti-Brn3a (1:100 dilution, #SC-8429; Santa Cruz Biotechnology, Santa Cruz, CA). Secondary antibodies were: goat anti-rabbit Alexa 488 (1:100 dilution, #A11034; Molecular Probes, Eugene, OR) and goat anti-mouse Alexa 546 (1:100 dilution, #A11030; Molecular Probes). Sections were deparaffinized in xylene and sequential alcohol rinses, then treated with 95°C Trilogy (CMX833-C; Cell Marque) antigen retrieval reagent. Tissue sections were subsequently blocked with Rodent Block M (RBM96; BioCare Medical, Concord, CA) for 30 minutes to reduce nonspecific binding. Slides were incubated overnight at 4°C in PBS containing primary antibodies, washed with PBS, incubated in antibody buffer containing secondary antibodies for 1 hour at room temperature, washed with PBS, and mounted onto glass coverslips using DAPI vectashield (Vector, Burlingame, CA). Images were collected on an inverted microscope (Axiovert 200M; Carl Zeiss Meditec, Oberkochen, Germany) running the Zeiss AxioVision 4.7.2 on PC (Zeiss Inc., Thornwood, NY). Confocal microscopy images were obtained in a Leica TCP SP5 spectral confocal microscope (Leica, Exton, PA; 63×, water-immersion, 1.2 NA objective). Cell counts were taken across the extent of each retinal section examined. The total anti-Brn3a cell counts and anti-calretinin cell counts of the Brn3b -/- and phenotypically wild-type mice were compared using a paired t-test. P < 0.05 was considered statistically significant.