Anti cd34 and anti pdgfrα antibody

IHC was performed according to the protocol provided by the anti-CD34 and anti-PDGFRα antibody manufacturer (Abcam, Cambridge, England). Briefly, after deparaffination, the sections (5 μm) were incubated in 3% H₂O₂/methanol for 15 min. After washing the sections in PBS (0.01 M pH 7.4) for 3 × 5 min, they were pre-treated using heat mediated antigen retrieval with 0.01 M sodium citrate buffer (pH 6.0) for 15 min, and then washed in PBS for 3 × 5 min. All following steps were carried out in a moist chamber. The sections were incubated in normal goat serum at room temperature for 15 min. After discarding the goat serum, the sections were incubated in the anti-CD34 and anti-PDGFRα antibody (Abcam, Cambridge, England) with diluted in PBS (1:200) at 4 °C overnight, respectively. After rinsing in PBS for 3 × 5 min, the sections were incubated with biotinylated anti-rabbit IgG at 37 °C for 30 min. Afterwards, they were detected using an HRP conjugated compact polymer system at 37 °C for 30 min. The 3,3-diaminobenzidin (DAB) was used as the chromogen. The colouration was terminated with PBS when positive cells were clearly visible. The sections were counterstained with haematoxylin and mounted. Finally, the sections were also photographed by using BM 2000 light microscopy.