Nitroblue tetrazolium nbt

1,1-diphenyl-2 picrylhydrazyl (DPPH), 2,2′-Azinobis (3-ethyl benzthiazoline-6-sulphonic acid) liquid substrate (ABTS), trolox, ascorbic acid (AA), hydrogen peroxide (H₂O₂), Folin-Ciocalteu phenol reagent, nitroblue tetrazolium (NBT), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), catechin, quercetin, orlistat were obtained from Sigma-Aldrich Chemicals (St Louis, MO, USA). Other chemicals of analytical grade were purchased from Merck Limited (Mumbai, India).

Purified STEC₀₂₆ and STEC₀₁₁₁ EspA, EspB, EspF, NleA and Tir recombinant proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane using a Mini Trans-Blot Electrophoretic Cell (Bio-Rad) as per the manufacturer’s instructions. The membranes were probed with either polyclonal sera (1:2500) from mice vaccinated with a pool of the corresponding STEC_O103 recombinant proteins or with rabbit polyclonal sera (1:2500) raised against STEC_O103 EspA, EspB, EspF, NleA and Tir. Alkaline phosphatase labeled goat anti-mouse or goat anti-rabbit IgG (KPL) antibodies were used as secondary antibodies (1:2000). The membranes were developed using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) salt according to the manufacturer’s instructions (Sigma).

Three terminal leaflets were appraised from each plant from three different positions (top, middle, and lower section), and six biological replicates per treatment were sampled for in situ histochemical localization of superoxide anion (O₂^•−) and hydrogen peroxide (H₂O₂) using nitro blue tetrazolium (NBT; Sigma–Aldrich, Darmstadt, Germany) and 3,3′-Diaminobenzidine (DAB; Sigma-Aldrich, Darmstadt, Germany), respectively. For O₂^•− histochemical staining, fresh leaflets were vacuum infiltrated, with 10 mM potassium phosphate buffer (pH 7.8) containing 0.1% NBT (w/v) [41 (link)], incubated for 20 min under light and then cleared with 0.15% trichloroacetic acid (w/v) in ethanol to chloroform 4:1 (v/v). O₂^•− was visualized as a purple coloration of NBT. For histochemical detection of H₂O₂, fresh leaflets were vacuum infiltrated with 0.1% DAB in 10 mM tris buffer (pH 7.8) and then incubated under light for one hour. Following staining, leaflets were cleared as described above and the intensity of brown color was estimated using the ImageJ image processing program (Fiji version; http://fiji.sc; access date 10 May 2021). A Chemi Imager 4000 digital imaging system (Alpha Innotech Corp., San Leandro, CA, USA) was used to measure the discoloration due to NBT or DAB staining.

2,2-Diphenyl-2-picrylhydrazyl (DPPH; Aldrich), FeCl₂·4H₂O (Fluka), trichloroacetic acid (Sigma), phenazine methosulfate (PMS; Sigma), nicotinamide adenine dinucleotide (NADH; Sigma), nitro blue tetrazolium (NBT; Sigma Aldrich), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma), butylated hydroxytoluene (BHT; Aldrich), stigmasterol (Sigma), caffeic acid (Sigma), wedelolactone (Sigma), and ethylenediaminetetraacetate (EDTA; Sigma) were purchased from Sigma Chemical Co. (St. Louis, MO). Chlorogenic acid was purchased from Acros Organics (Thermo Fisher Scientific Inc.). Ferrozine, ferric chloride (FeCl₃), and potassium ferricyanide (K₃Fe (CN)₆) were purchased from Showa Co., Ltd. (Tokyo, Japan). Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen), fetal bovine serum (FBS, Gibco), and penicillin-streptomycin were purchased from Gibco BRL (Life technology, Paisley, Scotland).

The aescin used in the study was amorphous aescin powder (CAS: 6805-41-0), isolated from the seeds of Aesculus hippocastanum L. (Horse chestnut seed), with known therapeutic activity: >95% triterpene glycosides (residual solvents: <5% of water, ethanol, and traces of methanol), and was a kind gift from the Chemical Industries Development Company (CID), Giza, Egypt. Thiopental sodium (batch number 1806554) and testosterone oenanthate (batch number 20601001) were obtained from EIPICO (10^th of Ramadan City, Egypt) and CID, respectively. Electrochemiluminescence immunoassay kits for testosterone (Elecsys Testosterone II; cat. No. 05200067) and luteinizing hormone (Elecsys LH; cat no. 11732234122) were purchased from Roche Diagnostics, Mannheim, Germany.
Mouse monoclonal antibodies against TNF-α, IL-1β, and TGF-β1 were purchased from Santa-Cruz Biotechnology (Heidelberg, Germany), while β-actin mouse monoclonal antibodies were from R&D SYSTEMS, Minneapolis, MN, USA. Alkaline phosphatase-tagged anti-mouse antibody, 5-bromo-4-chloro-3-indolyphosphate (BCIP), and nitro-blue tetrazolium (NBT) were purchased from Sigma-Aldrich, St. Louis, MO, USA. All other chemicals were of analytical grade.