Anti proinsulin

We fixed the mice, after exsanguination, in 4% paraformaldehyde by perfusion and isolated dorsal root ganglia (DRG) at L3–L5. Isolated thigh bones were decalcified in 10% EDTA for one week. DRG and thigh bones were cut into 10 μm-thick sections and incubated with anti-proinsulin (mouse monoclonal; Fitzgerald Industries International, Acton, MA, USA), anti-TNF-α antibody (goat polyclonal; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-microtubule-associated protein 2 (MAP2) antibody (rabbit polyclonal; Cell Signaling Technology, Danvers, MA, USA). Next we incubated the sections with species-matched fluorescence-labelled second antibodies, and observed the sections under a fluorescence or light microscope (Zeiss, Thornwood, NY, USA; Nikon, Tokyo, Japan). We counted 350–500 DRG neurons per mouse in at least three sections separated by 50 μm intervals. The number of immunopositive cells was normalised to the total number of neurons counted.

We isolated total BM cells from the femur, humerus and tibia of mice under anaesthesia. Mononuclear cells were isolated from total BM or peripheral blood cells using Ficoll-Paque (GE Healthcare Bio-Sciences, Uppsala, Sweden) and then incubated with anti-proinsulin (Fitzgerald Industries International) and anti-TNF-α antibody (Santa Cruz Biotechnology). Next we incubated the sections with species-matched fluorescence-labelled second antibodies. Data were collected using a FACS Calibur (BD Biosciences, San Jose, CA, USA).