H3410

Serially cut frozen slides were air-dried and fixed in prechilled 100% acetone). Slides were rinsed in distilled water and then washed in TBS-T. Endogenous peroxidase activity was quenched via incubation with 5% (v/v) H₂O₂ (Sigma-Aldrich, H3410) in dH₂O. After 30 minutes, the slides were washed and incubated in serum-free protein block (DAKO, X0909) for 30 minutes, after which excess liquid was removed. Serial sections were then incubated with 1:1000 (v/v) anti-MPO (mouse, monoclonal; Abcam, ab25989) or 1:1000 (v/v) anti-NE (rabbit, monoclonal; Abcam, ab131260) or 1:500 (v/v) anti-CitH3 (rabbit, monoclonal; Abcam, ab219407) for 1 hour in antibody diluent (bovine serum albumin [1% w/v], Sigma-Aldrich, A7906) and Triton-X100 (0.5% v/v, Sigma-Aldrich, 270733) in TBS-T. Slides were further washed in TBS-T and incubated with horse radish peroxidase (HRP)-coupled secondary antibody (anti-mouse, DAKO, K4001; anti-rabbit, K4003) for 30 minutes. Following washing in TBS-T, slides were incubated for 10 minutes with Opal690 fluorophore (PerkinElmer, FP1497) in tyramide signal amplification (TSA) buffer (PerkinElmer, FP1498, 1:50 v/v dilution in 1x plus amplification buffer) and subsequently washed. Specimens were incubated in a 1:800 (v/v) dilution of Spectral DAPI for 10 minutes, washed, and then cover-slipped with fluorescence mounting medium.

Catalase (CAT) activity was evaluated by spectrophotometry (240 nm), through the consumption of hydrogen peroxide (H₂O₂; Sigma–Aldrich Corporation, H3410) by measuring decreasing absorbance, whose rate of decomposition is straightly proportional to its activity. Superoxide dismutase (SOD) activity was determined through measures of oxidative pirogalol (Sigma–Aldrich Corporation, P0381). A colorful by-product based on oxidation of pirogalol was detected by spectrophotometry (420 nm, SP22, Bioespectro) (Fridovich, 1986 (link)).

Intracellular ROS was detected using a CM-H2DCFDA kit (General Oxidative Stress Indicator, Invitrogen, C6827). Samples were collected in cold HBSS (Gibco, 14175095) and kept on ice until the analysis with the BD LSR2 Cell analyzer. 5 µM dye was added 20 min before the read out on site. 100 µM hydrogen peroxide (Sigma-Aldrich, H3410) for 30 min was used as a positive control. Data were analyzed using Flowjo (BD Biosciences, v10).