Ab133579

After the last scan, two mice/cell line xenografts were sacrificed and tumors were collected for immunohistochemical (IHC) evaluation of the PSMA expression levels as previously reported^{28 (link)}. In short, sections were taken from the center of the tumor sample and stained using Haematoxylin/Eosin, incubated with a primary PSMA antibody (1:400, 2 h, ab133579, Abcam) and counterstained using Haematoxylin (Mayer). Sections were digitally scanned with a virtual scanning microscope (Olympus BX51, Olympus Belgium SA/NV, Berchem, Belgium) at high resolution (20 × magnification).

Tumor samples were obtained from the PC-3 bearing mice after micro-PET imaging (n = 5). Each specimen was formalin-fixed and paraffin-embedded for immuno-assaying. Tissue slices (3.0-mm thick) were dried in 74°C for 30 min and incubated with 3% H₂O₂ at room temperature for 10 min. Then sections were put into 0.01 M citric acid buffer for antigen retrieval and microwaved for 20 min. The tissue slices were stained with a rabbit anti-PSMA antibody (ab133579, dilution1:100, Abcam) for PSMA and rabbit anti-CD34 antibody (ab81289, dilution1:100) for CD34. They were incubated in 37°C incubator for 30 min, and then biotinylated anti-rabbit IgG was added onto tumor sections for another 30 min. The sections were also stained with DAB (diaminobezidin). Stained tissue sections were examined under a microscope.

After the last scan, two mice/cell line xenograft were sacrificed and tumors were collected for immunohistochemical (IHC) evaluation. Sections were taken from the centre of the tumor sample and stained using Haematoxylin/Eosin, incubated with a primary PSMA antibody (1:400, 2 h, ab133579, Abcam) and counterstained using Haematoxylin (Mayer). Sections were digitally scanned with a virtual scanning microscope (Olympus BX51, Olympus Belgium SA/NV, Berchem, Belgium) at high resolution (20 × magnification).

Cells were lysed in RIPA buffer, and proteins were collected and determined by BCA protein assay kit (Beyotime Biotechnology). 40 μL protein sample with 10 μL precleared AgroseA/G Beads was incubated for 1 h at 4°C. Another 100 μL precleared AgroseA/G Beads was blocked with 10 μL BSA (50 mg/mL) for 1 at 4°C. Blocked beads were collected and resuspend with lysis buffer. The blocked beads (10 μL) were added into protein sample and incubated with 5 μL PMSA (1:10, # ab133579, Abcam), ITGB4 (1:250, # ab182120, Abcam) or rabbit non-specific IgG antibodies overnight. 1 × SDS and 5 × protein sample was added into collected beads and boiled for 10 min, then 40 μg samples were subjected to SDS-PVDF for separating. PVDF membrane was blocked with 5% skimmed milk TBST, and incubated with PSMA (1:50,000), ITGB4 (1:1,000) and GAPDH (1:5,000, # ab179467, Abcam) antibodies at 4°C overnight. Following incubated with secondary anti-mouse/ribbit IgG (HRP) (1:5,000, # 7076/7074, CST) for 1 h. Proteins were detected with enhanced chemiluminescence (Thermo Fisher Scientific) and blot images were analyzed using the ImageJ.