Publication protocol
"Specimens detected with alkaline phosphatase were dewaxed and rehydrated, then incubated for 1 h with one of two blocking solutions. For FGF-10 detection, blocking was achieved with 5% normal rabbit serum in TBST [10 mM Tris·HCl (pH 7.5), 250 mM NaCl, and 0.3% (vol/vol) Tween 20]. For recognition of the FGF-10 receptor, blocking was completed with 1% (wt/vol) BSA (fraction V, Sigma) in TBST. Primary antibodies directed against human FGF-10 (catalog no. AF345) and the human FGF-10 receptor (catalog no. MAB665) were purchased from R&D Systems (Minneapolis, MN) as goat polyclonal and mouse monoclonal IgGs, respectively. The former was further affinity-purified using a previously described technique (3). After blocking, slides were incubated at room temperature for 4 h with either 1.67 μg/ml of the anti-FGF-10 IgG or 20 μg/ml of the receptor antibody, diluted in their respective blocking solutions. After a series of washes with TBST, sections were incubated at room temperature with secondary antibodies conjugated to alkaline phosphatase for 1 h. Secondary antibodies used were alkaline phosphatase-conjugated rabbit anti-goat IgG (3 μg/ml, catalog no. 305–055-045, Jackson ImmunoResearch, West Grove, PA) for FGF-10 detection and sheep anti-mouse IgG for detection of the FGF-10 receptor (1:100 dilution, catalog no. A3563, Sigma). Slides were then washed with TBST and rinsed with detection buffer at pH 9.5 (Roche Applied Science, Indianapolis, IN), followed by color development using substrates 4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate (Roche) in detection buffer. Images were acquired with a Leica DMR light microscope equipped with a Leica DC200 digital camera at image resolutions of 1,280 × 1,024 (6.7 μm/pixel).
For horseradish peroxidase-catalyzed reactions, dewaxed and rehydrated sections were subjected to heat-induced epitope retrieval by submersion in 1 liter of 10 mM citric acid (pH 6.0) and 10–16 min of microwaving. After cooling, sections were blocked for endogenous peroxidases by a 10-min incubation with 3% (vol/vol) hydrogen peroxide, washed with water, and assembled onto Sequenza coverplates (Thermo Scientific, Waltham, MA). After further blocking for endogenous avidin and biotin moities (DakoCytomation, Carpenteria, CA), a 1:10 dilution of normal rabbit serum (DakoCytomation) in TBS [50 mM Tris·HCl (pH 7.6), 150 mM NaCl] was applied to each slide for 5 min. Sections were incubated overnight at 4°C with one of the following primary mouse monoclonal antibodies in 0.1 ml TBS that contained 0.1% (wt/vol) BSA: anti-collagen type IV (3.13 μg/ml, Developmental Studies Hybridoma Bank at the University of Iowa, catalog no. M3F7), anti-K7 (1.1 μg/ml, catalog no. NCL-CK7-OVTL, Vision Biosystems/Novocastra, Norwell, MA), anti-K13 (1:20 dilution, catalog no. AB22685, Abcam), anti-K14 (0.5 μg/ml, catalog no. MCA890, Serotec, Raleigh, NC), anti-K17 (10 μg/ml, catalog no. C9179, Sigma), or anti-uroplakin III (1:50 dilution, mouse monoclonal IgG obtained from T. T. Sun, New York University School of Medicine). Slides were then rinsed three times with TBS and incubated with rabbit anti-mouse biotinylated secondary antibody (3.3 μg/ml, catalog no. E0354, DakoCytomation) at room temperature for 30 min, and rinsed three times again with TBS. StreptABComplex/HRP (DakoCytomation) was added to slides and incubated for 30 min. Slides were rinsed twice with TBS, once with ultrapure water, and treated with 1× Enhanced DAB Metal Substrate solution (Pierce, Rockford, IL) for 15 min. After two more rinses with water, sections were counterstained with a 1:1 dilution of Gill's formula hematoxylin (Vector Laboratories, Burlingame, CA), then dehydrated by a series of ethanol and xylene substitute washes. Images were captured as described above."
Full paper
Login or
join for free to view the full paper.