Immunohistochemistry
After being baked at 60 °C for 2 hours, the paraffin sections were
dewaxed and hydrated in turn, washed with PBS and incubated with 3%
hydrogen peroxide for 5-10 minutes (at room temperature) to eliminate
the activity of endogenous peroxidase. After antigen repair, goat serum
was added and incubated at room temperature for 15 minutes. The primary
antibody working solution containing TRPV1 (1:200), Kir6.1 (1:200),
SUR2B (1:200), and eNOS (1:200) was added to cover the tissue, and the
tissue was incubated at 4 °C overnight. After being rinsed, the
secondary antibody was diluted with PBS, added and incubated at room
temperature for 2 hours. Then, DBA reagent was used for color
development, the samples were stained again with hematoxylin and
subjected hydrochloric acid alcohol differentiation, dehydration,
transparency, and sealing with neutral gum. Then, the images were
observed and photographed under a microscope. The expression of various
proteins in endothelial cells was mainly observed. Brown–yellow
staining indicated positive expression, and the relative expression
level is represented by the optical density value. The images were
analyzed by Q-IMAGING software.