H e stain kit

Skin samples of the abdomen were collected from 6-week-old mice, fixed in 4% paraformaldehyde for 24 h, dehydrated in ascending series of alcohol, and then N-butanol, paraffin-embedded, and sectioned. The sections were then dewaxed by xylene, hydrated, and stained with HE Stain Kit (Beyotime, C0105M) or Masson’s Trichrome Stain Kit (Solarbio, G1340-7) according to the manual. The sections were then dehydrated through a gradient series of ethanol, cleared by xylene, sealed with neutral resin, and observed under a microscope (Olympus BX53, Tokyo, Japan).

The hall makers of VSMCs and macrophages were identified by immunofluorescent stains which were performed in accordance with our previous descriptions [16 (link)]. After animals were sacrificed by CO₂ suffocation, aortic roots were dissected and fixed with paraformaldehyde (4%, v/w). 5 μm paraffin tissue sections were made. For H&E stain, sections were washed by PBS and then processed with an H&E stain kit (Beyotime). The vessel lumen area (A1) and internal elastic lamina area (A2) were measured with ImageJ software (version 1.53e, NIH). The stenosis percentage was calculated as follows: (A2 − A1)/A2∗100%. Fixed cells or slides were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich). After blocking buffer (Abcam) incubation, slides were washed and treated with specific primary antibodies against inducible nitric oxide synthase (iNOS, 1 : 200, Abcam), myosin heavy polypeptide 11 (MYH11, 1 : 200, Abcam), RAGE (1 : 200, Abcam), and TLR4 (1 : 200, Abcam) at 4°C for 8 h. Slides were washed by PBS and incubated with secondary antibodies conjugated to Alexa Fluor 488 (Abcam) at 25°C for 20 min. Cell nuclei were stained with DAPI (Abcam). A fluorescence microscope (Nikon) was used to observe. The captured images were further analyzed by ImageJ software.

For hematoxylin–eosin (H&E) staining, hippocampus tissue of rats was harvested with caution, fixed in 4% paraformaldehyde (P6148, Sigma-Aldrich, USA), and finally embedded in pre-dissolved paraffin (1.07151, Supelco, Bellefonte, PA, USA). Samples were resected, stained by an H&E Stain Kit (C0105, Beyotime, Shanghai, China), and analyzed under an inverted light microscope (CKX53, Olympus, Tokyo, Japan) under × 100 and × 200 magnifications.