Elastica

Rabbits were sacrificed with an injection of phenobarbital sodium and xylazine hydrochloride. The aortic tree was isolated, opened longitudinally, fixed in 10% neutral buffered formalin, and stained with Sudan IV [15] (link). The area of the atherosclerotic lesion (Sudanophilic area) was measured using an image analysis software (Mitani Co., Tokyo, Japan) [15] (link).
For the microscopic quantification of the lesion area, the aortic arch was cut into 8–10 sections. Sections were embedded in paraffin and cut into 5-µm thick serial sections, stained with hematoxylin and eosin (H&E), and elastica van Gieson (EVG). For microscopic evaluation of the cellular components, serial paraffin sections were immunohistochemically stained with antibodies (Abs) against macrophages (MΦ) (Dako Inc. Carpinteria, CA), smooth muscle α-actin (Thermo Fisher Scientific Inc. Rockford, IL), matrix metalloproteinase-1 (MMP-1) and MMP-9 as previously described [16] (link). In order to evaluate the collagen contents, the sections were stained with Masson's trichrome staining as previously described [17] (link). To evaluate the cellular proliferation and death in the lesions, we performed TUNEL staining using the in situ cell death detection kits (Promega, Madison, WI) and immunohistolchemical staining using the Ab against PCNA (Bioss, Beijing, China). TUNEL and PCNA positive cells were counted as previously reported [18] (link).
To evaluate the plaque stability, we used frozen sections embedded in OCT compound and stained them for oil red O [11] (link) and Masson's trichrome, MΦ and smooth muscle cells (SMCs). The plaque vulnerability was calculated by dividing the sum of area of MΦ and extracellular lipid deposits by sum of area for SMCs and collagen fibers as previously described by others [18] (link).

The common iliac arteries and urinary bladders from each group were fixed in 10% neutral-buffered formalin, embedded in paraffin and cut into 5 μm sections. Slides were cleared with xylene, dehydrated and used for staining with Elastica–Masson (EM) stains. The common iliac arterial wall thickness was determined by averaging the wall thickness at four distinct locations in each sample using a microscope and cell Sens Dimension image analysis software (OLYMPUS, Tokyo, Japan) [37 (link)]. The bladder was processed for methylene blue staining and immunohistochemical staining. The number of MCs was counted in 5 consecutive fields (×400), and the mean values were used in the subsequent statistical analysis [10 (link)]. For immunohistochemical examinations of bladder tissue, sections were deparaffinized, and endogenous peroxidase was quenched with 0.3% H₂O₂. Nonspecific immunoglobulin G binding was blocked with 5% skim milk. Sections were incubated overnight at 4 °C with primary antibodies for anti-RAR2 (GR 324700-11, 1:200; Abcam, Cambridge, UK), anti-UP Ia (GR160579-1, 1:200; Abcam), anti-UP Ib (GR153380-5, 1:200; Abcam), anti-UP II (14277, 1:200; Proteintech, Rosemont, IL, USA) and anti-UP III (GR31809451, 1:200; Abcam). After rinsing three times in phosphate-buffered saline for 5 min, appropriate species-directed secondary antibodies (Signal Stain Boast IHC Detection Reagent; Cell Signaling Technology, Danvers, MA, USA) were applied to the sections at room temperature. When substances staining positive were located in the cytoplasm as dark-brown granules, these cells were judged as positive in the immunohistochemical evaluation. The percentage of UP-positive cells on the urothelium was assessed. The percentage of UP-positive cells in each rat was calculated in four randomly selected high-power fields (×400).