Goat anti rabbit igg hrp

The antibodies used in this study included rabbit anti-OPTN antibody (Cat. No. 10837-1-AP; ProteinTech Group, Inc., Chicago, IL, USA), mouse anti-FLAG M2 monoclonal antibody (F3165; Sigma-Aldrich), rabbit anti-GFP antibody (MBL International Corporation, Woburn, MA, USA), monoclonal anti-α-tubulin antibody (T8203; Sigma-Aldrich), goat anti-mouse IgG/HRP (P0447; DAKO, Tokyo, Japan), goat anti-rabbit IgG/HRP (P0448; DAKO), and rabbit anti-goat IgG/HRP (P0449; DAKO), and AlexaFluor488-conjugated donkey anti-mouse IgG (A21202; Invitrogen, Carlsbad, CA, USA).

Tissue sections were first incubated for 2 hours at 68°C; dewaxed with xylene, anhydrous ethanol, a gradient ethanol series and distilled water; and then boiled in acid buffer (pH of 6.0) for 2 min at a high temperature and pressure. Then, endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide. The sections were incubated with 5% normal goat serum for 30 min, and then ZNF24 antibody (11219-1-AP, Proteintech) and SLC7A5 antibody (28670-1-AP, Proteintech) were diluted at a ratio of 1:200 and incubated at 4 °C overnight. Goat anti-mouse IgG-HRP or Goat anti-rabbit IgG-HRP (ready-to-use, DAKO) was incubated at 37°C for 1h and then stained with DAB for 30s-60s.

Samples from the cortex, and inner and outer medulla were homogenized in buffer (0.3 mol/L sucrose, 0.25 mmol/L imidazole, 1 mmol/L EDTA containing protease inhibitors leupeptin (8.5 μmol/L), phenylmethylsulphonyl fluoride (PMSF) (1 mmol/L), and 1% SDS). They were centrifuged (5000 g for 15 min at 4°C) to remove cellular debris and total protein measured using BCA reagent (Pierce Chemical Co., Thermo Fisher Scientific, Rockford, IL). Laemmli buffer was added in the ratio of 1: 2. The samples were separated on 12% SDS premade TGX polyacrylamide gels (BioRad, Hercules, CA) and electroblotted onto Immobilon PDVF membranes (Millipore, Billerica, MA). Loading and transfer equivalence were assessed by staining with Ponceau S. The membranes were blocked with 5% nonfat milk in buffer (25 mmol/L tris, pH 7.6) containing 1% Tween 20 for 1 h and then incubated with the appropriate primary antibody. They were then washed, reacted with the secondary antibody (goat anti‐rabbit IgG‐HRP, DAKO), and visualized by chemiluminescence using Supersignal Pico West (Pierce Chemical Co., Thermo Fisher Scientific). X‐ray films were scanned using a BioRad (GS‐700) densitometer using Quantity One, version 4.3.2 (BioRad) software.