Bovine serum

Three eyes 1 week after laser irradiation or three normal eyes were collected and fixed by immersion in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Sections (4 μm thickness) were made through the optic disc and blocked with 1% bovine serum (Roche Diagnostics GmbH, Mannheim, Germany). The primary antibodies were against p-AMPK (1:100; Sigma-Aldrich) and neurofilament-L (a marker of nerve fibers; 1:100; Dako, Tokyo, Japan). The secondary antibodies were FITC-labeled or rhodamine-labeled antibodies (1:100; Cappel, Aurora, OH, USA). The sections were mounted on slides in a DAPI-containing medium under the cover of glass. The images were captured using a confocal microscopy system (Zen; Carl Zeiss QEC GmbH, Köln, Germany).

Three eyes 1 week after intravitreal injection of NR or three normal eyes were collected and fixed by immersion in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Sections were made through the optic disc and blocked with 1% bovine serum (Roche Diagnostics GmbH, Mannheim, Germany). The primary antibodies were against NRK1 (1:100; LifeSpan BioSciences), neurofilament-L (a marker of nerve fibers; 1:100; Dako, Tokyo, Japan), or Thy-1 (a marker of RGC; 1:50; Santa Cruz Biotechnology, TX). The secondary antibodies were FITC-labeled or rhodamine-labeled antibodies (1:100; Cappel, Aurora, OH). The sections were mounted on slides in DAPI-containing medium with cover glass.

Twelve hours after vitreous injection of NMDA or PBS, removed eyes of 10 rats were fixed by immersion in 4% paraformaldehyde for 24 h. After paraffin processing and sectioned, deparaffinized sections were blocked with 1% bovine serum (Roche Diagnostics GmbH, Mannheim, Germany) and incubated with anti-p-c-Jun antibody (Cell Signaling Technology, Inc.) or anti-Thy-1 antibody (Santa Cruz Biotechnology) diluted 1:100 in 1% BSA in PBS overnight. The secondary antibodies were FITC-labeled anti-rabbit antibody, or rhodamine-labeled anti-mouse antibody (Cappel, Aurora, OH, USA). Photomicrographs of the sections were obtained with a confocal microscope (LSM510, Carl Zeiss).