A mouse antihuman monoclonal antibody against EGFR (clone 2-18C9, #1492; Dako, Glostrup, Denmark), ready for use, and a rabbit monoclonal antibody against the pEGFR (Tyr 1173, #4407; Cell Signaling Technology, Beverly, MA, USA) was used at a dilution of 1 100.
Use a mouse or touch screen.
Proliferative activity of the tumours was assessed by using the mouse antihuman monoclonal Ki67 antibody (clone MIB-1) obtained by DAKO (Glostrup, Denmark).
Oestrogen receptor immunohistochemistry was carried out using Novocastra 6F11 mouse antihuman ER (Novocastra, Newcastle-Upon-Tyne, UK) with a manual protocol at a dilution of 1 : 50, with epitope retrieval carried out using EDTA at pH 8.0 with a microwave pressure cooker technique for 5 min.
Methods for immunohistochemical detection of proliferating cells by BrdU (using mouse anti-BrdU, diluted 1 20; Roche Molecular Biochemicals, Indianapolis, IN) and endothelial cells by CD31 (using mouse antihuman CD31, diluted 1 30; DAKO, Cambridgeshire, UK) were as described previously (11, 14).
To determine whether the human sIL-18Rα complex is present in the sera of healthy individuals, we prepared an anti-IL-18Rα mAb affinity column to isolate the complex using mouse antihuman IL-18Rα H44 mAb [ 30].
CD138+ subsets were isolated from the mononuclear cells fraction by using mouse antihuman CD138 antibodies (Miltenyi Biotech, Auburn, CA, USA) coupled to magnetic microbeads (Miltenyi Biotec), followed by magnetic column selection (magnetic-activated cell sorting, Miltenyi Biotec), as previously described (Matsui et al, 2004).
Immunohistochemistry was performed using a rabbit monoclonal antihuman ER antibody (clone SP1; Dako, Cambridgeshire, UK) and a polyclonal rabbit antihuman PR antibody (Dako).
Knockdown of the GnRH-I receptor protein was demonstrated with immune cytochemistry by using a monoclonal mouse anti-human GnRH-I receptor antibody and immunoblotting by using a polyclonal rabbit antihuman GnRH-I receptor antiserum (not shown; c.f. [ 23]).
Knockdown of the GnRH-I receptor protein was evaluated with immune cytochemistry by using a monoclonal mouse anti-human GnRH-I receptor antibody (clone A9E4; Research Diagnostics, Flanders, NJ, USA) and immunoblotting by using a polyclonal rabbit antihuman GnRH-I receptor antiserum (Peptide Research Laboratories, Heidelberg, Germany), as described previously [ 23].
