Collision Metastases of Breast and Rectal Carcinoma – A Possible Role for Chemokines Receptors Expression

Appendix 1. Material and Methods

Immunostaining was performed by the peroxidase-indirect-polymer method. Sections 3 μm thick were cut, unto Superfrost plus slides, from paraffin-embedded routine tissue blocks. The sections were de-waxed, rehydrated and subjected to epitope antigen retrieval (20 min, 94°C) with Target Retrieval Solution High pH 50x Envision™ Flex (Ref.: DM828) in a pre-treatment module PTlink (Dako, Code No. PT 10130).

After blocking nonspecific staining primary monoclonal mouse antibodies anti-human CCR6 (Clone 53103.111; R&D Systems ref.: MAB195, 1:40), CCR7 (Clone 150503; R&D Systems ref. MAB197, 1:20), Synaptophysin (Clone SY38; Dako ref. MO776, 1:20), anti-GCDFP-15 (Clone 23A3; Novocastra NCL-GCDFP15, 1:60) and polyclonal rabbit anti-human Chromogranin A (Dako ref. A0430, 1:9000), were incubated for 30 min at room temperature. Endogenous peroxidase was blocked using Peroxidase Blocking Reagent Envision™ (Ref.: SM801). A labelled polymer HRP anti-mouse/rabbit (EnVision™ Flex ref. SM802) was used for all immunohistochemistry and incubated at room temperature for 30 min. DAB (3,3′—diaminobenzidine) was used as chromogen and Mayer Haematoxylin for counterstaining. Positive controls: tonsil and colon adenocarcinoma were used for CCR6 and CCR7 and appendix, pancreas and sweat glands for Chromogranin A, Synaptophysin and GCDFP-15 respectively. For negative controls, primary antibodies were omitted during the staining.