2.11. Immunofluorescence

Primary tumors were double-stained using a whole-mount staining protocol for CD31 and NG2 (Liu et al., 2018; Li et al., 2016). Tissues were fixed in 4% paraformaldehyde for 24 h, paraffin-embedded, sectioned, dewaxed in xylene, and rehydrated through graded alcohols. Antigen retrieval was performed in citric acid buffer (pH = 6.0). Sections were blocked in 2% normal goat serum for 1 h and stained with primary antibodies at 4 °C overnight. Endothelial cells and pericytes of tumor vessels were identified by staining with combinations of two antibodies. Endothelial cells were labeled with rat monoclonal anti-CD31 (1:500), and pericytes were labeled with rabbit anti-mouse NG2 antibody (1:400). Tumor tissues were double-stained with anti-CD31 and anti-NG2 antibodies. The sections were then washed and incubated with rhodamine-conjugated goat anti-rat IgG (H + L) (1:50) or goat anti-rat IgG-FITC (1:200) for 40 min at room temperature. The level of pericyte coverage was presented as a percent of the length along CD31⁺ vessels. Images were photographed under a fluorescence microscope (Olympus, Japan) and analyzed using ImageJ software (Public domain).