Goat anti rabbit immunoglobulin g igg

The tumour tissues were embedded in paraffin, sectioned, and were subjected to xylene dewaxing. Citric acid was used for antigen retrieval. A 3% H₂O₂-methanol solution was used to block endogenous peroxidase for 15 min, followed by blocking with 5% bovine serum albumin for 20 min. The primary anti-cluster of differentiation (CD)31 polyclonal antibody (1:50, ab28364, Abcam, UK) was added dropwise at 37 °C and incubated for 2 h. Goat anti-rabbit immunoglobulin G (IgG; Proteintech, USA) labelled with horseradish peroxidase was incubated at 37 °C for 30 min. 3, 3-diaminobenzidine (Solarbio, Beijing, China) was used to develop colour, and haematoxylin was used for counterstaining; the sections were then dehydrated, rendered transparent, and cover-slipped with Permount mounting medium (Thermo Fisher Scientific, Inc., USA). The sections were observed under a × 100 optical microscope (Olympus, Japan) to detect the positive protein expression in the tumour tissue.
Microvascular density (MVD) quantitative analysis was performed according to a method proposed previously (Weidner et al. 1992 ). In each section, the number of microvessels stained with CD31 was counted in five fields, and the average value was used as the MVD value of the tumour tissue.