Biotinylated swine anti rabbit

Formalin-fixed tumour or liver tissues were paraffin-embedded and serial sections were cut by the Molecular Pathology Node, Cellular Pathology, Royal Victoria Infirmary, Newcastle-Upon-Tyne. H&E staining was also undertaken by the Molecular Pathology Node.
Formalin-fixed paraffin-embedded tumour and liver sections were dewaxed and hydrated. Endogenous peroxidase activity was blocked with hydrogen peroxide and antigen retrieval was achieved using 1 mM EDTA. Tissue was blocked using an Avidin/Biotin Blocking Kit (SP-2001, Vector Laboratories, Peterborough, U.K.) followed by 20% swine serum in PBS and then incubated with primary antibodies overnight at 4 °C. The following day, slides were washed and incubated with biotinylated swine anti-rabbit (E0353, Dako, U.K.) followed by Vectastain Elite ABC Reagent (PK7100, Vector Laboratories). Antigens were visualised using DAB peroxidase substrate kit (SK4001, Vector Laboratories) and counterstained with Mayer's haematoxylin. Immuno-stained cells were imaged using a DFC310 FX microscope (Leica Microsystems) and the images blinded (coded) prior to analysis by an independent party. At least five images per tissue at ×100 magnification (10× lens and 10× eye piece) were analysed using brown/blue pixel intensity using Adobe Photoshop.

Only double fluorescence was used for 5-HT2CR immunohistochemistry. The staining process was similar to that used for 5-HT2AR, apart from the use of different primary and secondary antibodies and different concentrations of antibodies and reagents. The sections were incubated in PBS containing 0.3% H₂O₂ for 30 min and then blocked in TBS-T with 2% BSA and 10% fetal bovine serum for 1 h. They were then incubated in rabbit anti-5-HT2CR (1:5000; cat#: ab32172, Abcam, Cambridge, UK) and goat anti-ChAT (1:100) in the blocking solution for 48 h at 4 °C. Next, the sections were incubated in biotinylated swine anti-rabbit (1:500; cat#: E0353, Dako, Glostrup, Denamrk) and ABC (1:100) for 1 h each in the blocking solution at RT and then incubated in biotinylated tyramide (1:10000; cat #: 6241, Tocris, Bristol, UK) in PBS and 0.005% H₂O₂ for 6 min, followed by 1 h of incubation each in donkey anti-goat IgG Alexa 488 (1:200; cat#: A11055, Invitrogen, Waltham, MA, USA) and streptavidin-conjugated Alexa 594 fluorophore (1:600; cat#: A11058, Invitrogen, Waltham, MA, USA) in the blocking solution at RT. The sections were thoroughly washed with PBS-T between the different steps. Finally, the sections were mounted onto the microscope slides and coverslipped.