Anti fap

NEOLP or EOLP lesions were isolated and cultured for OLP-MFs. 8 Primary OLP-MFs and 6 normal fibroblasts (NFs) were obtained according to methods described by a previous study (12 (link)). Cultured cells at passages 3-6 were used. OLP-MFs and NFs were examined using anti-cytokeratin (1:100; cat. no. AM06387SU-N; OriGene Technologies, Inc.), anti-vimentin (1:200; cat. no. ab92547; Abcam), anti-α-SMA (1:100; cat. no. ab5694; Abcam) and anti-FAP (1:100; cat. no. ab53066; Abcam) antibodies. Growth and viability of OLP-MFs were assessed using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) at 24, 48 and 72 h, according to the manufacturer's protocol. The cells were rinsed with PBS and fixed overnight in 3% glutaraldehyde at 4˚C. Subsequently, the samples were dehydrated using the following ethanol gradient: 30, 50, 70, 95 and 100%. The samples were then dehydrated with xylene, air-dried at room temperature and embed with epoxy resin. The specimens were coated with gold. The ultrastructure of OLP-MFs and NFs was compared using a transmission electron microscope (magnification, x1.2k) and scanning electron microscopy (magnification, x700), respectively. The images were processed using Adobe Photoshop CS6 (Version 13.0.1; Adobe Systems, Inc.).

On day 22, the tumors were excised and proteins were extracted using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Inc., Haimen, China) supplemented with protease inhibitor cocktail (100:1; Sigma-Aldrich). Tumor tissues were homogenized prior to western blot and PCR analysis in liquid nitrogen using a mortar and pestle. Total protein concentrations in the supernatant were determined via the Bicinchoninic Acid assay (P0013B; Beyotime Institute of Biotechnology, Inc.). A total of 30 µg protein was loaded per lane for western blot analysis. Protein samples were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). After blocking with 5% skimmed milk in TBS containing 0.1% Tween 20 (TBST) for 2 h at 37°C, the membranes were incubated with anti-FAP (1:500; 28244; Abcam) or anti-β-actin (1:1,000; sc-130657; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) primary polyclonal antibodies at 4°C overnight. After washing four times with TBST for 10 min, the membranes were incubated with the goat anti-rabbit HRP-conjugated secondary antibody (1:5,000; sc-2054; Santa Cruz Biotechnology, Inc.) for 1 h at 37°C. After washing with TBST as described above, the bands were visualized using enhanced chemiluminescence reagents (WBKLS0100 EMD Millipore).

FaDu tumors were excised and paraffin-embedded, followed by the standard dehydration process.^{41 (link)} The tumor samples were cut into 5-µm sections and stained with hematoxylin and eosin (H&E). The sections were dewaxed and antigen retrieval was performed in a sodium citrate buffer. The following antibodies were used: anti-FAP (Abcam) and anti-A27 (GenScript Corporation). Biotinylated secondary antibodies (goat anti-rabbit; Jackson ImmunoResearch Laboratories, Suffolk, UK) were used and detection was performed with Vectorstain Elite ABC reagent and Vector ImmPact DAB Peroxidase substrate (Vector Laboratories, Burlingame, CA).
DU145 tumors were excised at 36 dpi, followed by paraformaldehyde fixation, and then cut into 100-µm sections. The blood vessels and cell proliferation were detected with anti-CD31 antibody (BD Pharmingen, San Jose, CA) and anti-Ki67 antibody (BD Pharmingen), respectively.
The examination of tumor sections was conducted with an MZ16 FA fluorescence stereomicroscope (Leica, Buffalo Grove, IL) equipped with a digital charge-coupled device camera (Leica). Digital images (1,300 to 1,030-pixel images) were processed using Adobe Photoshop 7.0 software (San Jose, CA).