Nb600 636

The mouse eyeballs were fixed in Davidson’s fixation solution for 48 h for the paraffin section. Following the antigen retrieval with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) in a steam bath, three washes with phosphate-buffered saline (PBS) and blocking with 5% bovine serum albumin (BSA) in PBS, the sections were incubated with anti-PPARα (Novus #NB600-636; Centennial, CO), anti-TOMM20 (abcam #ab186735; Waltham, MA), or anti-nitrotyrosine (abcam #ab61392) antibodies overnight. After three washes with PBS, the slides were incubated with Alexa Fluor 488 AffiniPure donkey anti-rabbit IgG (Jackson ImmunoResearch #711-545-152; West Grove, PA) or Alexa Fluor 594 AffiniPure donkey anti-mouse IgG (Jackson ImmunoResearch #715-585-150). The slides were mounted with Vectashield mounting buffer containing DAPI (Vector Laboratories #H-1200; Newark, CA) and photographed under a Zeiss Microscope (Observer Z1; Pleasanton, CA).

Western blot analysis was performed as described previously (20 (link)). The equal amount of protein was resolved by SDS-PAGE and immunoblotted with primary antibodies for PPARα (Novus #NB600-636), TOMM20 (abcam #ab186735), PGC-1α (Novus #NBP1-04676), and β-actin (Sigma-Aldrich #A5441). Primary antibodies were then detected with HRP-conjugated secondary antibodies (Vector Laboratory #PI-1000 & PI-2000), and the band intensity was semiquantified by densitometry using ImageJ software and normalized by β-actin levels.