Vectastain elite abc reagent

The paraffin-embedded tissue sections (5 μm) were immunostained with E-cadherin, N-cadherin, vimentin, IGFBP2 and Ki-67 antibodies. Tissue sections were deparaffinized in xylene and rehydrated with ethanol. Tissue sections were then preincubated with 10% normal goat serum in PBS followed with incubation with primary antibody overnight at 4°C. Tissue sections were then stained with biotinylated secondary antibody (Vector lab, USA) for 1 hour at room temperature, followed by incubation with the Vectastain Elite ABC reagent (Vector lab, USA) for 30 min. The peroxidase reaction was developed with diaminobenzidine (DAB kit; Vector lab) and the slides were counterstained with hematoxylin (Sigma). The number of positive cells was counted in five high-power fields using a microscope (Olympus, Japan).

Tissue sections were de-paraffinized with Histo-Clear II (National Diagnostics, Atlanta, GA, USA) and then transferred through four changes of 100%, 96%, and 70% ethanol and water. Antigen retrieval was performed in a pressure cooker filled with sodium citrate buffer at pH 6.0. Endogenous peroxidase activity was quenched for 10 min with 3% hydrogen peroxide. Blocking was performed for 15 min with Avidin followed by 15 min with Biotin (Avidin/Biotin blocking kit , Vector Labs, Burlingame, CA, USA). Samples were washed with PBS and blocked with 5% horse serum for 30 min at room temperature to reduce nonspecific staining. After washing, primary antibodies to Wnt-11 (Bio-Techne R&D AF2647 at 1:200 and Genetex GTX105971 at 1:50) and β-Catenin (BD610154 at 1:250) were applied overnight at 4 °C. Sections were incubated with biotinylated secondary antibody (Vector Labs) for 30 min followed by Vectastain^® Elite ABC reagent (Vector Labs) for 30 min. Liquid diaminobenzidine (DAB) (DAKO) was used as a chromogenic agent for 1–2 min and sections were counterstained with Mayer’s hematoxylin. Images were taken on an AxioImager D1 light microscope (Carl Zeiss Iberia SL, Madrid, Spain).

BD Heparin Binding Plates (BD Biosciences) were used in all assays. HS, DS, and heparin (Iduron) were dissolved in PBS at concentrations of 0.05–12.8 μm based on approximate “average” disaccharide MWs. Plates were coated with GAG overnight at room temperature then washed with Wash Buffer (100 mm NaCl, 50 mm NaAc, 0.2% Tween20, pH 6.0), blocked with Wash Buffer +1% BSA (Sigma) for 90 min at 37 °C. Following incubation with 200 ng/ml CXCL12 in PBS for 90 min at 37 °C, wells were blocked with Wash Buffer/5% goat serum (Vector Labs) before adding 1:1000 polyclonal rabbit αhuman CXCL12 antibody (AbCam) for 1 h at room temperature. Wells were stained with biotinylated goat αrabbit IgG (Vector Labs,1:2000), amplified with Vectastain Elite ABC reagent (Vector Labs) and developed with OPD substrate (Sigma) as per manufacturer's instructions. Absorbance at 490 nm was measured using a Synergy HT Microplate Reader (BioTek).

The sections with TMAs were deparaffinized and rehydrated following a standard protocol. Next, the TMAs were incubated with anti-menin antibody (#ab92443) at 1:50 dilution, at 36 °C for 1 h. Sections were washed with PBS and the antigen-antibody complexes were further stained using Vectastain Elite ABC Reagent (Vector Laboratories). Finally, the sections were developed using DAB substrate (Vector Laboratories) for visualization. A section was counterstained with hematoxylin and eosin (H&E). All sections were mounted with di-n-butylphthalate-polystyrenexylene (DPX) and the slides were visualized with a BX41 Olympus inverted microscope equipped with a digital camera (Olympus DP71). Images were acquired at 20X magnification. Menin staining was scored as follows: no staining = 0, very weak and patchy perinuclear immunoreactivity = 1, moderately intense perinuclear linear immunoreactivity = 2, and intense, diffuse, and finely granular nuclear immunoreactivity with a more pronounced linear perinuclear pattern = 3. The fibrosis was identified and considered in this project when the tumor glands were embedded in more than 60% of a dense collagenous glassy fibrotic background. We also graded tumors from I to III, in accordance with recommendations from the College of American Pathologists.