Alexa fluor 555 goat anti guinea pig

RGCs were immunostained in whole-mounted retinas and in cryostat sections of the eye. The immunohistochemical techniques were performed at the same time for all species studied. The whole-mounted retinas were blocked with phosphate buffered saline, pH 7.4 (PBS) containing TX-100 (0.25%) overnight, prior to incubation in the primary antibody. The sections were washed twice with PBS-TX-100 for 10 min, and they were then incubated overnight with a primary guinea pig antibody or rabbit anti-RBPMS antibody (1:1000; Phosphosolutions, Aurora, CO, USA), and goat anti-Brn3a (1:1.000; Santa Cruz Biotechnology, Dallas, TX, USA) to detect RGCs, and primary mouse anti-Neurofilament 200 antibody (1:200; Vector Laboratories, Burlingame, CA, USA) to label neuronal axons. After two washes with PBS, antibody binding was detected for 1 h (5 h for whole-mount retinas) with Alexa Fluor 555 goat anti-guinea pig and Alexa Fluor 488 goat anti-mouse secondary antibodies, or Alexa Fluor 555 donkey anti-rabbit and Alexa Fluor 488 goat anti-goat secondary antibodies (Invitrogen, Carlsbad, CA, USA) diluted 1:1000 in PBS-BSA (1%). The sections were washed twice with PBS for 10 min and mounted with a coverslip in PBS:Glycerol (1:1).