Rabbit anti heme oxygenase 1

Antigen retrieval was carried out following depara nization. The sections were placed into a pH 6.0 citrate buffer and heated at boiling temperature with a pressure cooker for 5 min. The 3% H 2 O 2 was used for blocking endogenous peroxidase activity at room temperature for 10 min. The specimens were washed with 0.01 mol/L phosphate buffer saline (PBS, pH 7.4) (5 min×3 times) and incubated using primary antibodies diluted in 0.1 mol/L PBS with 0.25% Triton X-100 and 0.2 % gelatin (PBSGT, pH 7.4) at 4°C overnight, including rabbit anti-Nrf2 (1:250, Abcam, UK), rabbit anti-MnSOD (1:100, Abcam, UK), rabbit anti-Heme Oxygenase 1(HO-1) (1:150, Abcam, UK) and mouse anti-NQO1 (1:100, Abcam, UK). After washing in PBSGT, sections were incubated with Alexa Fluor 488/594-conjugated (1:1000, Abcam, Cambridge, UK) or HRP-conjugated (1:800, Cambridge, UK) secondary antibodies at room temperature for 1 h. Images were captured respectively using a uorescent or optical microscope (Olympus, Tokyo, Japan). According to published literatures, immunohistochemistry and immuno uorescence analysis were performed with computer image analysis system (ImageJ; National Institutes of Health) [29] (link). At least ve representative elds per animal and three different mice per group were taken for comparative analysis.