Amab90535

Protein extraction from cell lines was performed using RIPA buffer (5 nM EDTA, 150 mM NaCl, 1% Triton, 20 nM Tris pH 8 and 1:200 protein inhibitors). After Bradford colorimetric protein quantification samples were run on a SDS/PAGE acrylamide gel for 2 h and transferred to a PVDF membrane. Membranes were blocked for 1 h with 5% milk and incubated with the primary antibodies described above overnight and with the secondary antibodies -Goat anti-rabbit (Ref. P0448, dilution 1:2000, Agilent; Santa Clara, CA, USA) and rabbit anti-mouse (Ref. P0260, dilution 1:2000, Agilent; Santa Clara, CA, USA)- for 1 h at room temperature. Membranes were developed using Immobilon Immobilon Western Chemiluminiscent (Ref. WBKLS0100, Merck Millipore; Billerica, MA, USA). Membranes were finally stained with naphtol blue (Ref. N3393, Sigma-Aldrich; St. Louis, MO, USA) to detect all proteins transferred to the membranes in order to normalize the ADAR protein bands. The following antibodies were tested: ADAR1 (Ref. AMAb90535, dilution 1:100, Sigma-Aldrich; St. Louis, MO, USA) and ADAR2 (Ref. sc-73409, dilution 1:50, Santa Cruz; Heidelberg, Germany, EU).

A total of three TMAs were constructed as described previously^{47 (link)}. Paraffin-embedded TMA or individual sections of the samples were mounted on slides, deparaffined and rehydrateted. Antigen retrieval was done during 4 min at 115 °C (pH 6) and subsequently blocked with peroxidase (3%) for 5 min and incubated with the primary antibody for 1 h and 30 min at room temperature in a humidified chamber. After blocking for 30 min with EnVision secondary antibody (Ref. K5007-100 ml, Agilent; Santa Clara, CA, USA) slides were treated with DAB (Ref. K3468, Agilent; Santa Clara, CA, USA) reagent and counterstained with hematoxylin for 30 sec. Finally, a dehytratation step and DPX mounting were performed. Pictures were taken using the Olympus BX41 microscope (20X). The following antibodies were tested: ADAR1 (Ref. AMAb90535, dilution 1:100, Sigma-Aldrich; St. Louis, MO, USA) and ADAR2 (Ref. sc-73409, dilution 1:50, Santa Cruz; Heidelberg, Germany, EU). A pathologist evaluated the expression using two criteria: the intensity of staining (0, no staining; 1, weak intensity; 2, moderate intensity; 3, high intensity) and the percentage of endometrial epithelial stained cells (0–100). The product of the two scores yielded final values on a sacale ranging from 0 to 300.