Immunohistochemistry
From each group, six randomly selected samples underwent
deparaffinization using xylene and were rehydrated with a graded ethanol
series. Endogenous peroxidase was blocked with a 3% hydrogen peroxide
solution after washing. It was then buffered with sodium citrate to
facilitate antigen retrieval (10.2 mM, 20 min, 95°C). Using PBS
containing 10% (w/v) bovine serum albumin, the sample was blocked for
10 minutes, and then at 4°C with a target of cadherin 5 (1: 200), SOD1
(1: 100), CD34 (1: 100), CASP3 (1: 200), or VEGF (1: 200). After that,
the slide underwent incubation at room temperature for two hours with
the corresponding HRP-conjugated secondary antibody (1:1000). Following
washing, DAB dyeing, and counterstaining with hematoxylin was
accomplished. A microscope was utilized to collect images for further
analysis from at least six random fields of view (Olympus Corp., Japan).