Graft vein tissues were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin wax, and cross-sectioned into 4 μm sections. Tissue sections were placed on an electric heating plate at 60–70 °C for 3–4 h, followed by dewaxing using xylene for 15 min at room temperature (20–25 °C). Dewaxing was completed after two repetitions. The sections were subsequently hydrated in a gradient of decreasing ethanol and washed with tap water for 5 min to remove excess ethanol. Sections were then H&E and Masson stained to examine the structural changes and degree of hyperplasia of the graft vein. The stained sections were observed under a light microscope (magnification, ×100; Leica, Wetzlar, Germany). The thickness of the intima and media was measured using ImageJ, and the intima/media ratio was calculated simultaneously. Three different parts of each sample were randomly selected for measurement, and the average value was calculated.
For H&E staining (Shanghai Biyuntian Biotechnology Co., Ltd., cat. no. C0105S), the sections were stained with hematoxylin for 4–5 min and washed with tap water for 5 min. The sections were placed in a hydrochloric acid-ethanol fast differentiation solution (Shanghai Biyuntian Biotechnology Co., Ltd., cat. no. C0163S) for 10 s and washed with water for 5 min. The hydrated tissue sections were again immersed in an eosin staining solution for 3 min. The sections were subsequently dehydrated using graded increments of ethanol and coverslips were fixed with neutral gum.
For Masson staining (Beijing Solarbio Technology Co., Ltd., cat. no. G1340), sections were stained with Ponceau S dye for 5 min and washed with tap water for 5 min. The sections were incubated with 1% phosphotungstic acid solution for 5 min and then stained with aniline blue dye for 5 min. Subsequently, they were incubated with 1% glacial acetic acid in water for 1 min, dehydrated with graded increments of ethanol, and fixed with neutral gum.
For H&E staining (Shanghai Biyuntian Biotechnology Co., Ltd., cat. no. C0105S), the sections were stained with hematoxylin for 4–5 min and washed with tap water for 5 min. The sections were placed in a hydrochloric acid-ethanol fast differentiation solution (Shanghai Biyuntian Biotechnology Co., Ltd., cat. no. C0163S) for 10 s and washed with water for 5 min. The hydrated tissue sections were again immersed in an eosin staining solution for 3 min. The sections were subsequently dehydrated using graded increments of ethanol and coverslips were fixed with neutral gum.
For Masson staining (Beijing Solarbio Technology Co., Ltd., cat. no. G1340), sections were stained with Ponceau S dye for 5 min and washed with tap water for 5 min. The sections were incubated with 1% phosphotungstic acid solution for 5 min and then stained with aniline blue dye for 5 min. Subsequently, they were incubated with 1% glacial acetic acid in water for 1 min, dehydrated with graded increments of ethanol, and fixed with neutral gum.
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