Dylight 549 conjugated goat anti rabbit igg

Tissue slices were washed three times with PBS. Antigen retrieval was performed in a microwave oven for 10 min in 10 mM citrate buffer (pH 6.0). After blocking for 1 h with normal goat serum prepared in PBS, the slices were incubated overnight at 4 °C with a mouse anti-Iba1 monoclonal antibody (1:200; #MABN92, Millipore Corporation, Temecula, CA) or a rabbit anti-4-hydroxynonenal (4-HNE) polyclonal antibody (1:400; #HNE13-M, Alpha Diagnostic International, San Antonio, TX, USA). The slides were then washed three times for 5 min with PBS. The following secondary antibodies were used for 50 min incubations at 37 °C: DyLight 549-conjugated goat anti‐rabbit IgG (1:200; #A23320, Abbkine Scientific Co., Ltd., Wuhan, China) and DyLight 488-conjugated goat anti‐mouse IgG (1:200; #A23210, Abbkine Scientific Co., Ltd., Wuhan, China). After washing and mounting, the sections were analyzed with an Olympus FV3000 confocal laser scanning microscope.

Immunofluorescence analysis for nitro-tyrosine was used to detect the hepatic endogenous RNS level, and WGA was used to mark the hepatocyte membranes. Briefly, liver frozen sections were permeated by 0.1% Triton X-100, followed by being blocked with 10% goat serum at RT for 1 h. Then, the sections were incubated with anti-nitro-tyrosine (1:200) and an anti-WGA (1:400) primary antibody at 4 °C overnight, followed with incubation with Dylight549-conjugated goat anti-rabbit IgG (#A23320, Abbkine, Wuhan, China) at RT for 1 h. The nuclei were then stained with DAPI and the slides were observed under a fluorescence microscope (Leica, Bensheim, Germany). An amount of 10% fresh liver homogenate was prepared in ice-cold phosphate-buffered saline (PBS). Subsequently, hepatic MDA (A003-1) contents, enzymatic activity of hepatic CAT (A007-1-1) and GPx (A005-1) were detected using corresponding commercial test kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Hepatic ROS levels were detected using a tissue ROS detection kit (#HR8821) obtained from Baiaolaibo Company (Beijing, China). All detections were performed according to the manufacturers’ instructions.