Rhodamine labeled peanut agglutinin pna

Three-week-old and 22-week-old mouse eyes were fixed with 1% paraformaldehyde (PFA) in 0.1M PB at room temperature (RT) for 1 hour (h). Eyecups were then prepared, cryoprotected in increasing concentrations of sucrose from 5% to 20% in 0.1 M PB, embedded in a 20% sucrose/OCT compound (Sakura Finetek USA, Torrance, CA) mix at 2:1, and quick frozen. Retinal sections were cut at 10μm and placed on Superfrost slides. The retinal sections of 3-week mice were pre-incubated in 10% horse serum for 1 h at RT, and then incubated with rhodamine labeled peanut agglutinin (PNA) (1:1,000; Vector Laboratory, Burlingame, CA) in 3% horse serum for 1 h at RT. After the incubation, sections were mounted with VECTASHIELD Mounting Medium with DAPI (Vector Laboratory). The retinal sections of 22-week mice were preincubated in 10% horse serum for 1 h at RT, and then incubated with an antibody against rhodopsin (1:200; Gene Tex, Irvine, CA) overnight at 4°C. After incubation with Alexa Fluor 488-conjugated anti-mouse IgG (1:500; Invitrogen, Carlsbad, CA) and rhodamine labeled PNA (1:1,000) for 1 h at RT, sections were mounted with VECTASHIELD Mounting Medium with DAPI. Images were taken on the LSM 510 Meta laser-scanning microscope (Carl Zeiss, Thornwood, NY).