Digital microscope camera

Mice were anaesthetized via the inhalation of isoflurane and euthanized by cervical dislocation. Kidneys were removed and fixed in 10% formalin. Specimens were embedded in paraffin and sliced into 2–3 μm in thickness. Subsequently, the kidney tissues were stained with Hematoxylin-eosin (H&E stain). The images were captured using a Nikon Digital Camera Microscope (Nikon, Tokyo, Japan).

Immunohistochemistry (IHC) was performed manually or automatically with an autostainer (BenchMark XT, Ventana Medical Systems Inc., Tucson, AZ, USA). For the manual protocol, paraffin sections were first deparaffinized and rehydrated in ethanol/water solutions. Epitopes on tissue were then retrieved with Heat-Induced Epitope Retrieval (HIER) in citrate buffer (0.01 M, pH 6.0). For blocking endogenous peroxidase activity, sections were treated with 3% hydrogen peroxide for 30 min in the dark. To reduce non-specific primary antibody binding, Blocking Buffer (DAKO) was used for one hour at room temperature. Sections were then incubated with primary antibodies at 4 °C overnight. Rabbit anti-mouse COX-2, anti-human cPLA2, and anti-8-OHdG polyclonal antibodies were purchased from Santa Cruz Biotechnology (SC-1747-R, SC-7891, and SC-139586, Santa Cruz, CA, USA). Afterward, staining was detected with a DAKO polymer system. For image acquisitions, three random high-power magnification fields were obtained in each sample by a Nikon Digital Camera Microscope (Nikon, Tokyo, Japan). All slides were reviewed by a blinded pathologist (Dr. Shih-Hao Liu), and the area percentage of staining in a 200× power magnification field were analyzed by NIH ImageJ software (Version 1.47, Bethesda, MD, USA).

Tissue sections of human arterial rings were evaluated quantitatively and qualitatively. The histological characterizations were assessed by hematoxylin and eosin stain. A Nikon Digital Camera Microscope (Nikon, Tokyo, Japan) was used for image capture. For tissue staining of arterial ring sections, we generally followed the introductions of the manufaculturer’s protocol: (1) von Kossa staining (CVK-1-IFU, ScyTek Laboratories, Logan, UT, USA.) to detect calcified lesions; (2) 8-hydroxy-2’-deoxyguanosine (8-OHdG) (Bs-1278R, Bioss Antibodies, Woburn, MA, USA. 1:200 dilution) for oxidative stress related vascular injury; (3) RUNX2 (Sc-101145, Biotechnology, Santa Cruz, CA, USA. 1:200 dilution) as an osteoblast-specific transcription factor; (4) ALP (Ab108337, Abcam, Cambridge, UK. 1:200 dilution) as a bone formation marker. The details of the quantitative method for IHC images were proposed previously [3 (link)]. In brief, three random high-resolution fields in each specimen were used for quantitative analyses via ImageJ version 1.48v image analysis software (National Institutes of Health, Bethesda, MD, USA).