O dianisidine dihydrochloride d3252

Soleus and EDL muscles from BLAJ and WT animals (n = 6 each) were homogenized in physiological buffer (1:50 mass:volume) and then 0.14 µg/µL amyloglucosidase (A1602, Sigma-Aldrich) was added. All sample tubes were heated at 70℃ for 30 min and 60 µL glacial acetic acid added, followed by two units of peroxidase-glucose oxidase (PGO) enzyme (P7119, Sigma-Aldrich, Saint Louis, MO, USA) which had been prepared by dissolving one capsule in 1 mL o-Dianisidine/dihydrochloride (D3252, Sigma-Aldrich) and made to 50 mL volume with ddH₂O (then kept in dark according to manufacturer’s instructions). Extracts were heated overnight at 55 ℃, following which 200 µL 4 M KOH was added and then 240 µL of PGO solution. A series of glucose standards were prepared using serial dilutions of a 1 mg/mL stock solution over the range 15–500 µg/mL glucose with a reagent blank of ddH₂O and treated as per the samples from the PGO step onwards. After 5–10 min and within 30 min, the absorbance of samples and standards were read on a UV-VIS spectrophotometer at 450 nm and the glucose content of samples determined relative to the glucose calibration curve.

Except where specified, all chemicals were obtained from Sigma-Aldrich (St Louis, MO, USA). Protease inhibitor cocktail was from Roche Diagnostics Corp. (Mannheim, Germany). ECL detection reagent was from GE Healthcare (Waukesha, WI, USA). Anti-Ctr1 ab123105, anti-Sod1 ab13499 were from Abcam (Cambridge, UK). Recombinant anti-PrP^C humanized Fab D18 ABR-0D18 was purchased from InPro Biotechnology (South San Francisco, CA, USA). Monoclonal anti-PrP SHA31 A03213 was from BertinPharma (Montigny le Bretonneux, France). Anti-TfR1 13-6800 was from Invitrogen (Paisley, UK). Anti-β-Tubulin III T2200, monoclonal anti-β-Actin Peroxidase (AC-15) A3854, anti-Steap3 AV43515, anti-Atp7a AV33797, anti-human Ferritin F5012 were from Sigma-Aldrich. Anti-Ccs (FL-274) sc-20141, anti-FtH (H-53) sc-25617 were from Santa Cruz Biotechnology (CA, USA). Anti-Cp 611488 was from BD Transduction Laboratories (Milan, Italy). Anti-Tf GTX21223 was from GeneTex (Texas, USA). Anti-Fpn1 NBP1-21502 was from Novus Biologicals (Littleton, CO, USA). The anti-Atp7b N-WNPD#1 was kindly provided by Prof. S. Lutsenko, Johns Hopkins University, Baltimore, MD, USA. O-dianisidine dihydrochloride D3252 was from Sigma-Aldrich. The ELISA kit for Hepdicin SEB979Mu was from USCN (UK).