Nitroblue tetrazolium

Protein damage was evaluated according to the nitroblue tetrazolium (NBT) reduction test, which involved the addition of nitroblue tetrazolium (Sigma-Aldrich) and 2 M glycine (pH 10) according to the method described by Virella et al. [20 (link)]. The absorbance was read at 530 nm. The results are expressed as nmol formazan/mg protein. Protein carbonylation in the lipoproteins was determined by treatment with 2, 4-dinitrophenylhydrazine (DNPH). DNPH reacts with protein carbonylated (PC) derivatives to form stable hydrazones, which exhibit an absorption peak at 370 nm [21 (link)]. A molar extinction coefficient of 21 × 10³ M⁻¹ cm⁻¹ was used to quantify PC content. Values are expressed as nmol PC/mg protein [22 (link)]. Dityrosine (DT) levels were determined using a fluorometric assay with 320 nm excitation and 405 nm emission [23 (link)]. The results are expressed as nmol DT/mg protein.

Diaphorase Activity. nNOS activity was evaluated by nicotinamide adenine dinucleotide phosphate-tetrazolium reductase staining (NADPH-TR) as described previously [20 (link)]. Sections of frozen tissue were fixed in freshly prepared 4% paraformaldehyde before they were incubated with NADPH-TR solution (nitroblue tetrazolium 0.2 mM (Sigma) and β-NADPH 1 mM (Sigma) in Tris buffer 0.2 M with 0.25% triton X-100, pH 7.3, sterile filtered) for 60 min at 37°C, washed in Tris buffer 0.02 M pH 7.4, and mounted in aqueous mounting medium (Vector Laboratories, VWR, Herlev, Denmark).
NADH Oxidase Activity. Nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) stains were performed according to standard procedures. Briefly, frozen sections were incubated with NADH-TR solution (nitroblue tetrazolium 2 mM (Sigma) and NADH 1 mM (Sigma) in Tris buffer 0.02 M pH 7.4, sterile filtered) for 45 min at 37°C and washed in Tris buffer 0.02 M pH 7.4 and H₂O followed by 10 min incubation with calcium chloride in 4% formaldehyde. The sections were washed in H₂O and fixed in acetone before aqueous mounting.

Muscle tissue was embedded in Tissue‐Tek OCT Compound (Sakura), and 7 μm thick serial sections for staining were cut using a cryomicrotome. To analyse the NADH dehydrogenase activity, we incubated the sections in 0.9 mM NADH and 1.5 mM nitro blue tetrazolium (Sigma) in 3.5 mM phosphate buffer (pH 7.4) for 30 min. To analyse the succinate dehydrogenase (SDH) activity, we incubated the sections for 1 h in 50 μM sodium succinate and 0.3 mM nitro blue tetrazolium in 114 mM phosphate buffer containing K‐EGTA (Sigma). Myosin heavy chain (Myh) immunostaining of muscle tissue sections was performed in the order of fixation, permeation, and incubation with primary antibodies against MyhI, MyhIIa, MyhIIb (Developmental Studies Hybridoma Bank), and laminin (Abcam). For Myh immunostaining, MyhIIa (BF‐32, 1:200), MyhIIb (BF‐F3, 1:200) (Developmental Studies Hybridoma Bank), and laminin (ab11575, 1:200; Abcam) antibodies were used. The images were captured and processed with a Nikon ECLIPSE TE‐2000 U inverted microscope using nis‐elements f software (Nikon), and the myofibre area was measured using imagej software. For muscle histology, the cryosections were stained with Mayer's hematoxylin and eosin (BBC Biomedical). The images were captured using a Nikon ECLIPS TE‐2000 U inverted microscope or tissue faxs imaging software (TissueGnostics).