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Table 1 Reagents and conditions of the immunohistochemical detection of COX-2 in formalin-fixed paraffin-embedded breast cancer lesions

From: Stromal, rather than epithelial cyclooxygenase-2 (COX-2) expression is associated with overall survival of breast cancer patients

  Ab1 Ab2 Ab3
Antigen retrieval heating up in 0.01 M citrate buffer pH 6.0 in a microwave oven (650 W) for 10 minutes
Blocking serum* 1.5% normal goat serum 1.5% normal horse serum 1.5% normal rabbit serum
Type of primary antibody rabbit monoclonal antibody (SP21 clone, RM-1921, Thermo Fisher Scientific, Waltham, Massachusetts, USA) mouse monoclonal antibody (CX229 clone, 160112, Cayman Chemical, Ann Arbor, Michigan, USA) goat polyclonal antibody (N-20, sc-1746, Santa Cruz Biotechnology, Santa Cruz, California, USA)
Antibody dilution (v/v in PBS pH 7.4) 1:100 1:200 1:500
Incubation period overnight at 4°C, in a humid chamber
Antibody detection system* Vectastain Elite ABC Kits appropriate for each type of primary antibodies
Peroxidase substrate 0.05% 3,3′ diaminobenzidine (DAB, Sigma-Aldrich, St. Louis, Missouri, USA) with 0.01% H2O2 and 0.06% NiCl2 (Sigma-Aldrich) in Tris–HCl buffer, pH 7.4
Counterstaining* nuclear red
  1. *(Vector Laboratories, Burlingame, California, USA).