Streptavidin peroxidase

The oxidation of NcADF cysteines was evaluated by alkylation using biotin-iodoacetamide, as described (Hill et al., 2009 (link)). NcADF was reduced by TCEP and then desalinized (see DTNB assay). Subsequently, the recombinant protein was oxidized with NCT or H₂O₂ for 30 min at room temperature. The excess of NCT and H₂O₂ were inactivated with 12 mM histidine/methionine and 2 U catalase, respectively, for 20 min. After inactivation, the protein solutions were incubated with 0.5 µM biotin-iodoacetamide (bio-IAM) for 20 min at room temperature. Bio-IAM was quenched by the addition of 30 mM β-mercaptoethanol (Gibco Thermo-Fisher Scientific). Afterward, the reactions were dried in the vacuum concentrator and the contents were suspended in a non-reductant sample buffer at a final concentration of 25 µM NcADF. The samples were resolved by SDS-PAGE and transferred to PVDF membranes, which were stained with DB-71 (0,008% w/v Direct Blue 71, 10% acetic acid, 40% ethanol). The alkylation of cysteines was detected by streptavidin-peroxidase (1:20,000; Thermo-Fisher Scientific) and observed after luminol incubation in radiographic films.

The immunohistochemical analysis of the kidney, heart, and aorta was done according to the methods described by Ogaly et al. [37 (link)]. The tissue sections were deparaffinized, rehydrated, and pretreated with 10 mM citrate buffer for antigenic retrieval. Sections were incubated for two hours at 4°C in a humidified chamber with one of the following primary antibodies: rabbit polyclonal anti-eNOS antibody diluted at 1 : 50 (Santa Cruz Biotechnology, USA) and the monoclonal anti-iNOS antibody diluted at 1 : 25 (Santa Cruz Biotechnology, USA). The tissue sections were incubated with a biotinylated goat anti rabbit and mouse antibody (Thermo Scientific, USA), streptavidin peroxidase (Thermo Scientific, USA), and 3,3′-diaminobenzidine tetrahydrochloride (DAB, Sigma). The slides were counterstained with Mayer's hematoxylin then dehydrated and mounted. Primary antibodies were replaced by PBS for negative controls. The stained sections were analyzed by Leica Qwin 500 Image Analyzer (Leica, Cambridge, England). In each field, the immunopositive area (dark brown) was recorded. Percentage of the positive stained area (%) was calculated as the mean of 10 fields/slide.

For immunohistochemistry, slides were deparaffinized, serially rehydrated and, after the appropriate antigen retrieval procedure, stained with monoclonal mouse anti-human Ki67 (M7240, Dako), followed by the appropriate secondary antibody. Immunoreactive antigens were detected using streptavidin peroxidase (Thermo Scientific) and the DAB Chromogen System (Dako). After chromogen incubation, slides were counterstained in hematoxylin (BioOptica) and images were acquired by Leica DMRD optical microscope (Leica). The percentage of KI67-positive cells was evaluated on digital images of 4–6 tumors or lungs per group (4–6 × 200 microscopic fields per sample); clear brown nuclei were regarded as positive cells and the percentage of labeling index (number of positive cells/total cells × 100) was calculated for each field, by two pathologists, independently.

Detection of CPDs, 6‐4PPs and DewarPPs in sunlight‐exposed DNA samples was achieved by ELISA using TDM‐2, 64M‐2 and DEM‐1 monoclonal antibodies, respectively (26, 27), with some modifications. Briefly, 96‐well polystyrene flat‐bottom microplates (Thermo Scientific, nontreated, clear, Cat. No. 260895), precoated with 0.0001% protamine sulfate, were coated in quadruplicate with heat‐denatured sample DNA (10 ng/well for CPDs, 200 ng/well for 6‐4PPs, 500 ng/well for DewarPPs). After blocking with 2% fetal bovine serum, each class of dimeric photolesions was detected with TDM‐2 (1/1000), 64M‐2 (1/1000) or DEM‐1 (1/10 000), followed by goat antimouse IgG (H + L) conjugated to biotin (1/2000; Fitzgerald, Acton, MA, 43R‐1334) and then streptavidin‐peroxidase (1/10 000; Thermo Fisher, Cat. No. 43‐4323). After treatment with o‐phenylenediamine (OPD) and H₂O₂, the absorbance of colored products derived from OPD was measured at 492 nm.

caspase-3, e-NOS, and TNFR activity was investigated in corneal sections using immunohistochemical methods. The primary antibodies were caspase-3 (Bioss, Woburn, MA, USA; Cat. No:bs-0081R), e-NOS (Bioss, Cat. No:bs-0163R), TNFR1 (Bioss, Cat. No:bs-2941R). All primary antibodies were diluted with 1:100 Antibody Diluent Reagent Solution (Life Technologies, Carlsbad, CA, USA; Cat. No: 003118). The block solution, streptavidin peroxidase solution, and secondary antibody were used as kits (Cat. No: TP-125-HL). Sections that were 4–5 μm thick were deparaffinized and dehydrated. The sections were then incubated with 3% hydrogen peroxide, Ultra-V Block (ThermoFisher, Waltham, MA, USA), primary antibodies (Bioss), secondary antibody (ThermoFisher), and streptavidin peroxidase (ThermoFisher). Marking with DAB solution (Vectorlab, Burlingame, CA, USA) and nuclear staining with hematoxylin were performed. In this study, a previously reported semiquantitative scoring system was used to evaluate immunoreactivity [20 (link)]. Immunostaining intensity was scored as follows: 0, negative; 1, weak; 2, moderate; and 3, strong. The percentages of positively stained cells were calculated and scored as follows: 0–4% = 1, 5–19% = 2, 20–39% = 3, 40–59% = 4, 60–79% = 5, and 80–100% = 6. Finally, multiplicative quick scores were calculated [21 (link)].

IHC was performed on mouse lung tissue to examine KI67 expression. Lung sections were deparaffinized and rehydrate to water. Antigens were retrieved by heating the sections in BorgDecloaketRTU (Biocare medical, Concord, CA) for 10 minutes at 95C. After antigen retrieval, non-specific binding was blocked using Ultra V Block (UltraVision Detection System, Thermoscientific, Fremont, CA) for 10 minutes, then slides were incubated in humidity chamber with primary antibody from ABCAM (Cambridge, MA) at 1:4000 dilution for 1 hour at room temperature. As negative controls, adjacent slides were incubated in normal rabbit serum. After two buffer washes, slides were incubated for 10 minutes with biotinylated Goat Anti-polyvalent (UltraVision Detection System, Thermo Scientific), followed by a buffer wash and incubated for 10 minute in Streptavidin Peroxidase (UltraVision Detection System from Thermo scientific, Fremont, CA). After a buffer wash, staining of interest was developed with DAB chromagen (Dako, Carpinteria, CA) and counterstained with Fast Green (Sigma St. Louis, MO). Slides were then dehydrated and mounted with Cytoseal (Thermo Scientific, Fremont, CA). Quantification of immunohistochemistry staining was done as previously described.[11 (link)]