Anti fgf10 antibody

Mouse lung sections were stained with hematoxylin-eosin to determine the extent of injury. The lung injury was graded in a blinded manner as described previously [29 (link)]: 0 = no injury; 1 = area of lung injury less than 25%; 2 = area of lung injury between 25 and 50%; 3 = area of lung injury between 50 and 75%; and 4 = area of lung injury more than 75%. Immunohistochemistry was performed with an anti-prosurfactant protein C (proSP-C) antibody (MeckMillipore, Darmstadt, Germany) or an anti-FGF10 antibody (ABclonal, Boston, USA). Sections were covered with a DAB tetrahydroxychloride solution and then were counterstained with hematoxylin. The numbers of SP-C-positive cells were counted in 6 random fields (× 200), and the results were expressed as the average number of SP-C-positive cells/lung nucleated cells from 3 or more animals in each group. Scoring of FGF10 protein expression was determined by the hybrid scoring system (H-score) [30 (link)]. The intensity of the staining was classified into 4 grades: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining, and then score was multiplied by the percent of positive cells in the area. All the scores were performed in a blinded manner.

Samples of primary lesions and bone metastasis were fixed in formalin and embedded in paraffin and analyzed by immunohistochemical analysis. The 4 μm sections were deparaffinized and rehydrated, and their endogenous peroxidase activity was inhibited with 0.3% H₂O₂ methanol. After being blocked with the 5% normal goat serum for 1 hour at room temperature, the slides were incubated with the following primary antibodies at 4°C overnight: anti-TGF-α polyclonal antibody (ImmunoWay, Plano, TX, 1 : 50), anti-CD68 antibody (Abcam, Cambridge, UK; 1 : 200), anti-IL-6 antibody (ImmunoWay, Plano, TX, 1 : 50), anti-VEGF antibody (ImmunoWay, Plano, TX, 1 : 100), anti-FGF10 antibody (ABclonal, Boston, USA, 1 : 100), anti-FGF17 antibody (ImmunoWay, Plano, TX, 1 : 100), and anti-TGF-β1 antibody (ABclonal, Boston, USA, 1 : 100). Following incubation with biotinylated secondary antibodies, the streptavidin-biotin complex/horseradish peroxidase was applied. Finally, the immunoreaction signal was developed with DAB staining, and the slides were counterstained in hematoxylin.
The stained tissue sections were reviewed under a light microscope (Nikon ECLIPSE Ni-U, Tokyo, Japan). The numbers of TGF-α and CD68 positive cells were counted in 5 random fields (400x) of paraneoplastic tissue of each tissue section, respectively.