To further explore the therapeutic mechanism of the PDCP-NP, immunohistochemical staining of glioma was performed for the detection of Ki67 or CD31 after the different treatments. The antibodies used were rabbit monoclonal antibody to Ki67 (1:200, Abcam, Cambridge, UK) or CD31 (1:200, Abcam, Cambridge, UK). Paraffin-embedded tumors were cut into 5-mm-thick sections, de-paraffinized in xylene series and hydrated in distilled water. Antigen retrieval was obtained by the citrate buffer and washed with PBS. Samples were blocked with 3% H2O2 for 5 min, followed by treatment with primary antibodies for 2 h at 37 °C, HRP-secondary antibody for 30 min at 37 °C and were then stained with DAB and counterstained with hematoxylin. The stained sections of the brain were examined and recorded with an optical microscope. The number of stained cells was evaluated by manual counting of four random microscopic fields (400× original magnification) per subject.
Immunofluorescence staining of the CSC markers (CD133) were also performed on deparaffinized sections. Sections were stained with anti CD133 followed by goat anti-rabbit IgG Alexa FluorVR(488) (ab150083, 1:1500, Abcam, Cambridge, UK). The nuclei were stained using DAPI (Beyotime, Nantong, China). Angiogenesis was observed by confocal laser microscopy (A1 PLUS, Japan).
Immunofluorescence staining of the CSC markers (CD133) were also performed on deparaffinized sections. Sections were stained with anti CD133 followed by goat anti-rabbit IgG Alexa FluorVR(488) (ab150083, 1:1500, Abcam, Cambridge, UK). The nuclei were stained using DAPI (Beyotime, Nantong, China). Angiogenesis was observed by confocal laser microscopy (A1 PLUS, Japan).