Ab66157

Macroscopic examination of removed tissues was performed, and suprarenal abdominal aortas were used for histological analysis. Tissue segments were submerged in 4% paraformaldehyde for fixation, embedded in paraffin, and sectioned (5 μm). Sections of abdominal aorta (5 μm thick) were deparaffinized and rehydrated, then stained with elastin–Van Gieson (EVG) to assess the integrity of elastin layers. To detect target protein expression, primary antibodies against CD68 (1:500; ab53444, Abcam, Shanghai, China), NF-κBp65 (1:500; ab32536, Abcam, Shanghai, China), MMP-12 (1:500; ab66157, Abcam, Shanghai, China), and tumor necrosis factor (TNF)-α (1:200; ab6671, Abcam, Shanghai, China) were used. Nuclei were stained with 4′,6-diamidino-2-phenylindole or 3′,3-diaminobenzidine. Identical camera settings were used for all images.

For immunohistological analysis, two consecutive lung tissue sections were prepared. Examinations were done in rats exposed to either clean air, 3.0 mg/m³ CeO₂ NM-212 or 50.0 mg/m³ BaSO₄ NM-220 for one, 28 or 90 days. Lungs were fixed in 10% formalin and trimmed based on the work of Kittel et al. [86 (link)]. To detect the presence of lactoperoxidase (Lpo, rabbit polyclonal, Acris, NBP1-87010) and matrixmetalloproteinase 12 (Mmp12, rabbit polyclonal, Abcam, ab66157) antibodies directed against those markers were applied as described by Rittinghausen et al. [87 (link)]. Immunohistochemically stained lung sections were counterstained with hematoxylin and digitalized using slide scanner (MiraxScanner, Zeiss, Germany) and quantified using image analyzing software (ZeissZen, Zeiss, Germany). Quantified Lpo and Mmp12 levels were statistically evaluated using Kruskal–Wallis–ANOVA with Mann–Whitney U-test as post hoc analysis.