2.18. Biotin-switch assay

S-nitrosylation protein was analyzed in liver sections from young and old WT and TMBIM6^−/− mice. In situ detection of S-nitrosylated protein was performed using a modified biotin switch method. Briefly, after deparaffinization and rehydration of the liver sections, the sections were washed with 1xPBS (3 times) containing 0.4 mM EDTA and 0.04 mM neocuproine. Free thiol groups were blocked with 20 mM MMTS (Pierce) in PBS containing 0.4 mM EDTA, 0.04 mM neocuproine, and 2.5% SDS for 30 min. The blocking solution was removed and washed with 1xPBS (3 times) (composition as in the first step). Sections were then incubated with 20 mM sodium ascorbate (in PBS) for 15 min to reduce S-nitrosylated proteins. No sodium ascorbate was added to the negative control sections. Newly reduced cysteine residues were then labeled with 0.25 mg/mL biotin-HPDP (in PBS) for 30 min. Excess biotin was removed by washing 3 times with 1XPBS (composition as in the first step). Sections were then incubated at 4 °C/overnight with a fluorophore-tagged streptavidin Alexa Fluor 568 and IRE1α antibody (sc-390960) conjugated to Alexa Fluor 488 to visualize biotinylated proteins, followed by washing 3 times with 1XPBS. Cell nuclei were counterstained with DAPI. Images were acquired using a confocal laser scanning microscope. The entire procedure was carefully performed under protection from direct light.