WB: 1:500-1:1,000
IHC-P: 1:50-1:200
mIHC: 1:3,000
Fig1: Western blot analysis of FAP on Hela cell lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1704-23, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.
Fig2: Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue using anti-FAP antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1704-23, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Fig3: Fluorescence multiplex immunohistochemical analysis of the human pancreatic carcinoma (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD31 (M1511-8, green), anti-α-SMA (ET1607-53, red) and anti-FAP (ET1704-23, yellow) on human pancreatic carcinoma. Panel B: anti- CD31 stained on the endothelial cells. Panel C: anti-α-SMA stained on cancer-associated fibroblasts and smooth muscle cells. Panel D: anti-FAP stained on the cancer-associated fibroblasts. HRP Conjugated UltraPolymer Goat Polyclonal Antibody HA1119/HA1120 was used as a secondary antibody. The immunostaining was performed with the Sequential Immuno-staining Kit (IRISKit™MH010101, www.luminiris.cn). The section was incubated in three rounds of staining: in the order of M1511-8 (1/5000 dilution), ET1704-23 (1/1000 dilution), and ET1607-53 (1/3000 dilution) for 20 mins at room temperature. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 30 mins at 95C. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Nikon ECLIPSE Ni-E microscope.
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