Horseradish peroxidase hrp

Fluorescent ISH was performed as previously described (Watakabe et al., 2006 (link)) with some modifications. Riboprobes incorporating digoxigenin (DIG) and fluorescein (FL) were hybridised overnight. After washing, FL- and DIG-labelled probes were each detected in different ways. For detection of the DIG probes, the sections were incubated with an anti-DIG antibody conjugated with horse radish peroxidase (HRP) (1/500, Roche Diagnostics) for 6 hr at room temperature. After washing in TNTx (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Triton X-100) three times for 5 min, the sections were treated with 1:100 diluted TSA-Plus (DNP) reagents (Perkin Elmer) for 20 min. After washing in TNTx 3 × 10 min, the sections were incubated for 2 hr at room temperature with an anti-DNP antibody conjugated with Alexa 488 (1/500, Invitrogen). After quenching HRP activity and washing, the sections were incubated for 2 hr at room temperature with an anti-FL antibody conjugated with HRP (1/500, Roche Diagnostics) followed by reaction with TSA biotin reagents (Perkin Elmer) and visualisation with streptavidin conjugated with Alexa594 (Invitrogen).

Purified parasites were lysed in SDS-PAGE sample buffer. SDS-PAGE was performed with proteins from 10⁶ parasites per lane, and parasites were separated with 12% acrylamide gels. Proteins transferred on a polyvinylidene difluoride (PVDF) membrane were detected with rat anti-HA monoclonal antibody (MAb) 3F10 conjugated with horseradish peroxidase (HRP) (Roche), anti-GRA1 mouse MAb 92.10 B (33 (link)), and anti-GFP rabbit MAb (Life Technologies). Anti-mouse or -rabbit IgG conjugated with HRP (GE Healthcare Life Science, Little Chalfont, United Kingdom) was used as a secondary antibody.

The production of extracellular and intracellular reactive oxygen species (ROS) was measured in a chemiluminescence (CL)-based system [39 (link)] as CL activity in a ClarioStar plate reader (BMG Labtech) for family A or Biolumat LB 9505 (Berthold Co., Wildbad, Germany) for family B. The assay was performed in a white 96-well microtiter plate (volume 200 μl) or polypropylene tubes, respectively. For extracellular ROS measurements, neutrophils (100 μl, 5x10⁶ cells/ml) were mixed with IsoLuminol (5.64 μM, Sigma-Aldrich) and horseradish peroxidase (HRP, 0.2 U/ml, Roche Diagnostics, Basel, Switzerland). For intracellular measurements, neutrophils (100 μl, 5x10⁶ cells/ml) were mixed with Luminol (5.64 μM, Sigma-Aldrich), superoxide dismutase (SOD, f0.5 U/ml, Worthington Biochemical Corp., Lakewood, US-NJ) and Catalase (20 U/ml, Worthington). After mixing, the samples were pre-incubated for 5 min at 37°C. A background base line was then established before adding PMA (50 nM, Sigma-Aldrich) to the wells. The ROS production was measured for a total of 20 min or until the bulk of the reaction had concluded.

HFF cells that had been infected with T. gondii bradyzoites induced by 3 days of differentiation were harvested, centrifuged at 10,000 × g, and lysed in Laemmli buffer. The samples were resolved on a 4% to 15% polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). The membrane was blocked with 5% BSA–Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at room temperature before being probed with 20C3 MAb or anti-HA rat monoclonal antibody conjugated with horseradish peroxidase (HRP; Roche) at 1:500 for 1 h at room temperature. Following 20C3 MAb primary antibody incubation, the membrane was washed three times with TBST and then incubated with sheep anti-mouse antibody conjugated with HRP (GE Healthcare) for 1 h at room temperature. The membrane was then washed and scanned using an Odyssey imaging system (Li-COR).

Glass micropipettes were filled with a 4% solution of Horseradish Peroxidase (HRP, Roche) in 0.05 M Tris and 0.2 M KCl at pH 7.9 and then beveled to impedances between 40 and 88 MΩ (mean 72 MΩ +/− 12). Extracellular receptive fields (RFs) were hand-plotted and classified S or C, simple or complex (Martin and Whitteridge 1984b (link)). In successful attempts to impale the neuron its receptive field was checked to be sure that the extracellular receptive field belonged to the actual neuron and HRP was then iontophoresed into the cell (Martin and Whitteridge 1984a (link)).

Analytical-grade diethyl ether, n-butanol (Merck, VWRI, Leuven, Belgium), methanol, and ethyl acetate (Chem-Lab, Zedelgem, Belgium) were used in the extraction procedure. Sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl₂), ammonium acetate, potassium hydroxide, sodium hydroxide, acetic acid, ethanol, hydrogen peroxide (H₂O₂), dimethyl sulfoxide (DMSO), ethylene diamine tetra-acetic acid, disodium salt (EDTA-Na₂H₂), trypan blue, and tween-20 were supplied by Merck (VWRI, Leuven, Belgium). Paranitrophenyl phosphate, bis(N-methylacridinium) nitrate (lucigenin), phorbol 12-myristate 13-acetate (PMA), Percoll, sodium carbonate, and gallic acid were all purchased from Sigma (St. Louis, USA). Catechin and quercetin were from ChromaDex (LGC Standard, France). Amplex Red was purchased from Molecular Probes (Invitrogen, Merelbeke, Belgium). Bovine serum albumine (BSA) and horseradish peroxidase (HRP) were supplied by Roche (Germany). 2′,7′-Dichlorofluorescin-diacetate (DCFH-DA) was obtained from Eastman Kodak (Rochester, NY, USA). All the solutions were prepared with deoxygenated MilliQ water or ultrapure water (Easy Pure UV purification system, Barnested/Thermolyne, Dubuque, USA).