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The slides were incubated with primary antibodies targeting α6-integrin (1 100; Santa Cruz, Heidelberg, Germany), mPGES-1 (1:50; Thermo Scientific, Waltham, MA, USA) and EGFR (1 100; Cell Signalling, Leiden, The Netherlands) followed by chromogenic visualization using Immunoperoxidase Secondary Detection System kits (Chemicon, Billerica, MA, USA).

At the ultrastructural level, NPY-immunoreactivity was specifically detected by chromogenic visualization of DAB within the cytoplasm of fusiform and round cell bodies.

Immunoblotting was performed by electroblotting proteins from gels on to PVDF membranes, followed by immunoreactive polypeptide visualization using an alkaline-phosphatase-conjugated secondary Ab (antibody) with chromogenic detection [ 13].

Phosphorylated substrate was quantified by chromogenic detection using horseradish peroxidase-conjugated anti-phosphotyrosine antibody.

Peptides, identified by their position on the membrane, targeted by the protein of interest are visualized using bioluminescent or chromogenic substrates.

The Ab reactivity was revealed either by a chromogenic reaction using 3,3′diaminobenzidine tetrahydrochloride (DAB) and H2O2 as substrates or by the ECL plus system (Amersham).

For chromogenic visualization, biotinylated secondary antibodies, avidin boitin complex (ABC kit; Vector) and diaminobenzidine were used.

For chromogenic visualization of GFP expression, a mouse monoclonal antibody against GFP (sc-9996; Santa Cruz Biotechnology, Santa Cruz, CA) was used in conjunction with the MOM Kit (PK2200; Vector Labs, Burlingame, CA) for antigen detection [ 15, 28].

Chromogenic visualization was carried out applying the glucose oxidase-diaminobenzidine method described by Zaborszky and Heimer (1989).

Tissue sections were then washed in PBS and treated with streptavidin peroxidase (Lab Vision) for 10 minutes at room temperature followed by chromogenic development of immunoreactivity using 3-amino-9-ethylcarbazole (AEC kit, catalog number TA-125-SA; Lab Vision).

Gene amplification was evaluated by chromogenic in situ hybridization using Zymed Spot-Light EGFR and HER2 amplification probe.