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

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