4 hne

Paraffin-embedded 5-μm testicular sections were deparaffinized and rehydrated. Endogenous peroxidase was inactivated using 0.3% hydrogen peroxide and antigen retrieval was performed using Tris-EDTA buffer (Tris 10 mM, EDTA 1 mM, pH 9.0). Subsequently, sections were incubated with primary antibody against 4-hydroxynonenal (4-HNE, 1:1000, cat # HNE11-S, Alpha Diagnostic International) at 4 °C overnight. Sections were then incubated with anti-rat IgG horseradish peroxidase antibody (1:500, cat#7074, Cell Signaling Technology) for 1 h at room temperature, followed by a diaminobenzidine reaction, and finally counterstained with hematoxylin. Sections were examined under an Olympus BX43 microscope and photographed. Staining intensities of 4-HNE were analyzed using Image-Pro Plus software.

Paraffin-embedded liver sections (5 μm thick) were stained with primary antibodies using Dako Antibody Dilution solution (Dako, Carpinteria, CA). The primary antibodies used in these experiments were: rabbit anti-4-hydroxynonenal (4-HNE; Alpha Diagnostics, San Antonio, TX; 1:1000, 60 min, room temperature), mouse anti-human alpha-smooth muscle actin (αSma; abcam, Cambridge, MA; 1:200, 10 min, room temperature), goat anti-mouse TIM-1/KIM-1/HAVCR (R&D systems, Minneapolis, MN; 2 mm/ml, 10 min, room temperature), and rat anti-mouse CD68 (CD68; AbD serotec, Raleigh, NC; 1:200, overnight, 4°C). We used Dako EnVision System (Dako, Carpinteria, CA) HRP kit for HNE and αSma, Goat IgG HRP-conjugated antibody (R&D systems, Minneapolis, MN; 1:100, 10min, room temperature) as secondary antibody for KIM-1, and VECTASTAIN ABC Kit (Rat; Vector, Burlingame, CA) for CD68. Dako Liquid DAB+ Substrate chromogen System (Dako, Carpinteria, CA) was employed for visualization. Slides were counterstained with filtered Mayer’s hematoxylin (Sigma) for 5 min. Quantitative analysis was performed using Image-Pro Premier 9.1 (Media Cybernetics, Silver Spring, MD) at 100× magnification. For α-SMA and 4-HNE, five fields of Liver and Kidney tissue were randomly selected to calculate percent of positively stained area.

Fixed renal samples (whole kidney) were ethanol dehydrated, embedded in paraffin and sliced into 5 μm sections for periodic acid Schiff (PAS) staining using standard protocols [19 ]. Glomerular volume and mesangial expansion degree were determined from PAS-stained sections. Digital images of random glomeruli were obtained and analyzed by two independent observers. Mesangial matrix expansion was defined by increased amounts of PAS positive material in the mesangial region. The mesangial matrix expansion area was determined from the mean area of each glomerulus.
Paraffin sections were also stained with Picro Sirius Red for quantitative analysis of fibrosis [19 ]. Tissue containing collagen stains red while normal tissue stains green.
For immunohistochemical staining, kidney sections were incubated with primary antibodies to 4 -hydroxynonenal (4-HNE, Alpha Diagnostic International) or fibronectin (Abcam) overnight at 4 °C. Sections were then washed with PBS containing 0.1% Triton and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After the final wash with PBS/triton, sections were incubated with diaminobenzidine to visualize antigen-antibody complexes. ImageJ software was used for all quantitative measurements and subsequent calculations [20 (link)]