*2.4. Immunohistochemistry (IHC)*

Procedures utilized for fixation, O.C.T. embedment, and sectioning of mouse eyes were as described in detail previously by Ramachandra Rao et al. [16]. In brief, eyes were immersion fixed overnight in phosphate-buffered saline (PBS) containing freshly prepared paraformaldehyde (4% v/v), appropriately cryopreserved, embedded in O.C.T., and cryosectioning was performed on a Leica Model CM3050 S Cryostat (Leica Biosystems, Wetzlar, Germany). Retinal sections were first "blocked" with 0.1% BSA, 0.5% serum (species corresponding to secondary antibody host) in Tris-buffered saline containing 0.1% Tween-20 (TBST), then incubated for 1 h at room temperature with a rabbit polyclonal antibody against glial fibrillary acidic protein (GFAP;, DAKO/Agilent, Santa Clara, CA, USA; 1:500 dilution in TBST) and a mouse monoclonal antibody against the C-terminal epitope of opsin (1D4; Novus Biologicals, Littleton, CO, USA; 1:500 dilution in TBST), followed by incubation with fluor-conjugated secondary antibodies (AlexaFluor®-488 conjugated anti-mouse IgG, AlexaFluor®-568 conjugated anti-rabbit IgG; Thermo Fisher Scientific, Waltham, MA, USA; 1:500 dilution in TBST). Sections were then counterstained with DAPI and cover slipped with anti-fade mounting medium (Vectashield®; Vector Laboratories, Burlingame, CA, USA) and viewed with a Leica TCS SPEII DMI4000 scanning laser confocal microscope (Leica Biosystems). Images were captured using a 40X oil immersion (RI 1.518) objective under normal laser intensity (10% of laser power source), arbitrary gain (850 V) and offset (–0.5) values, to optimize signal-to-noise ratio. Digital images were captured and stored as TIFF files on a PC computer.
