Ho 1 rabbit

Total proteins extracts (20–40 µg) were separated on 4–12% Bis-Tris CriterionTM XT Precast Gel (Bio-Rad, Marne-La-Coquette, France) and transferred to an Immobilon PVDF membrane (Millipore, Molsheim, France). TLR-4 (mouse, 1/500; Abcam, Paris, France), HO-1-rabbit (rabbit, 1∶250; Abcam), COX-2 (rabbit, 1∶1 000; Abcam), NF-κB p65 (rabbit, 1∶5000; Cell Signaling, Danvers, USA), phospho-NF-κB p65 (Ser536, mouse, 1∶5000; Cell signaling) and β-actin (1∶8 000) primary antibodies (Abcam, Paris, France) were incubated overnight at 4°C. Anti-mouse or anti-rabbit (1/8 000, Sigma-Aldrich) horseradish peroxidase (HRP)-conjugated secondary antibody was incubated for 1 h at room temperature and developed by the LuminataTM Forte Western HRP substrate (Millipore, Molsheim, France) with Chemidoc XRS (Bio-Rad, Marne-La-Coquette, France).

Specific antigens in kidney, liver and spleen sections were detected using the Vectastain Elite ABC System (VectorLabs, Orton Southgate, UK), according to manufacturer’s instructions. The following primary mouse-specific antibodies were used: ATP7A, rabbit, polyclonal (Abcam, Cambridge, UK); ATP7B, rabbit, polyclonal to ATP7B C-terminal region (Abcam, Cambridge, UK); ceruloplasmin, rabbit, polyclonal (Abcam, Cambridge, UK); CTR1, rabbit, polyclonal to SLC31A1/CTR1 (Abcam, Cambridge, UK); F4/80, rat, monoclonal (AbDSerotec, Kidlington, UK); HO-1, rabbit, polyclonal (Abcam, Cambridge, UK); SOD1, rabbit, polyclonal (Abcam, Cambridge, UK). The secondary antibodies were biotinylated horse anti-rabbit IgG (H+L) (VectorLabs, Orton Southgate, UK), or goat anti-rat IgG2b:horse radish peroxidase conjugate (AbDSerotec, Kidlington, UK) with 3,3′-diaminobenzidine as the substrate (VectorLabs, Orton Southgate, UK). Images are representative of at least two replicates from at least four independent biological replicates. Brown colour indicates positive reaction. Blue colour indicates no staining.
For fluorescent detection of ATP7B, the secondary antibody used was goat anti-rabbit Alexa Fluor 647 (IgG H&L) conjugate (Abcam, Cambridge, UK). The staining was performed in the dark, after which the sections were mounted using VectaShield Mounting Medium with DAPI (VectorLabs, Orton Southgate, UK).