Extravidin peroxidase e2886

Formalin-fixed and paraffin-embedded tumor and spheroid sections (3 µm) were dewaxed using Roticlear (Carl Roth, Karlsruhe, Germany) and rehydrated in a graded series of ethanol. Antigen retrieval was performed in 10 mmol/L citrate buffer pH 6 intermittently heated to 100 °C in 5 min intervals. Washing was performed using 0.05 mol/L Tris-buffered saline pH 8 containing 0.5% (v/v) Tween-20 (TBS-T). Endogenous peroxidase was quenched using 3% H₂O₂ in TBS-T. Endogenous avidin and biotin were blocked using a commercially available avidin/biotin quenching system (Agilent, Santa Clara, CA, USA). Non-specific binding sites were blocked using 10% fetal bovine serum (v/v) in TBS-T. COX-2 was detected using the primary antibody ab15191 (Abcam, Cambridge, UK). Isotype controls were incubated with non-specific rabbit IgG ab27478 (Abcam). Specific binding was detected using the biotinylated secondary antibody 111-065-003 (Dianova, Hamburg, Germany) and ExtrAvidin-peroxidase E2886 (Sigma-Aldrich, St. Louis, MO, USA) followed by staining with 3-amino-9-ethylcarbazole substrate (Sigma-Aldrich). Tumor sections were counterstained with Meyer’s hematoxylin, mounted with Kaiser’s glycerol gelatin (Carl Roth), and imaged using the AXIO Imager A1 microscope (Carl Zeiss, Oberkochen, Germany).

Tissue sections were dewaxed in RotiHistol (ROTH) and re-hydrated in a graded series of ethanol (100, 96, 85, 70, 50% (v/v), H₂O). Antigen retrieval was performed in boiling 10 mmol/L citrate buffer (pH 6.0). Endogenous peroxidase was quenched with 3% (v/v) H₂O in Tris-bufferd saline containing 0.1% Tween 20 (TBS-T). Endogenous biotin was blocked using a commercial biotin blocking system (DAKO). Non-specific binding was blocked using 10% (w/v) fetal bovine serum in TBS-T. GHS-R1a was detected using the primary antibody ab134152 (ABCAM). Negative controls were incubated with blocking solution only. Specific binding was detected using the biotinylated secondary antibody 111-065-003 (1:200; DIANOVA) and ExtrAvidin peroxidase E2886 (1:50; SIGMA-ALDRICH). Sections were stained with 3,3′ diaminobenzidine, counterstained with eosin, and imaged using the AXIO Imager A1 microscope (CARL ZEISS).