Horseradish peroxidase conjugated rabbit anti mouse igg

To detect polymorphonuclear cells in kidney and liver, immunohistochemistry was performed on 5- μm tissue cryosections. Sections were fixated for 10 min using acetone. Next, sections were stained with HIS-48 mAb (supernatant, two times diluted) using an indirect immunoperoxidase technique. Endogenous peroxidase was blocked using H ₂O₂ 0.01% in phosphate-buffered saline for 30 mins. After thorough washing, sections were incubated with horseradish peroxidase-conjugated rabbit anti-mouse IgG as a secondary antibody for 30 mins, followed by goat anti-rabbit IgG as a tertiary antibody for 30 mins (both from Dako, Glostrup, Denmark). The reaction was developed using 9-amino-ethylcarbazole as chromogen and H ₂O₂ as substrate. Sections were counterstained using Mayer hematoxylin solution (Merck, Darmstadt, Germany). Negative antibody controls were performed. Localization of immunohistochemical staining was assessed by light microscopy. For each tissue section, positive cells per field were counted in 10 microscopic fields of the tissue at 40x magnification. Results were presented as number of positive cells per glomerulus in the kidney and number of positive cells per field in the liver.

This was assessed by measuring 3-nitrotyrosine (3-NT) formation using Western blot. Tissue samples (70 mg), taken from the ischaemic myocardium within 2 min of reperfusion, were prepared as described previously (Kiss et al., 2010 (link)). The formation of 3-NT was assessed from 25 μg of total protein loaded onto SDS-PAGE gel (10%) and transferred to PVDF membrane. Mouse monoclonal anti-nitrotyrosine was used as primary antibody (diluted to 1:3000; Chemicon, Millipore, USA), and horseradish peroxidase-conjugated rabbit anti-mouse IgG (diluted to 1:1000, Dakocytomation, Denmark) was used as a secondary antibody. The blot was developed with an enhanced chemiluminescence kit (ECL Plus, GE Healthcare, UK), exposed to X-ray film and scanned. The intensity of the 3-NT bands was determined using Image J software, and expressed in percentage of the sham-operated animals. Equal loading of the samples was controlled by Coomassie Brilliant Blue staining, and normalized for total protein. Protein samples, isolated from four dogs in each experimental group, were used for western blot. The measurements were repeated three times in each dog, and the results were averaged. Data obtained from the individual dogs within a group were also averaged, and these means served for the comparison among the groups.

Serum was prepared from blood obtained from mice at day 0, day 18, day 33 and day 63. Antibody titres were measured using enzyme linked immunosorbent assay (ELISA) as per methods described in Chua et al. (2011) (link). Briefly, ELISA plates (Nunc, Thermo Scientific) were coated overnight with 5 µg/ml protein diluted in PBSN₃ and blocked with BSA₁₀PBS for 2 h at room temperature. Plates were washed with PBS containing 0.05% Tween-20 (PBST). Neat sera were sequentially diluted in BSA₅PBST and incubated at room temperature for 6 h. Bound antibody was detected by adding horse radish peroxidase conjugated rabbit anti-mouse IgG (Dako, Glostrup, Denmark) or rat anti-mouse IgM, IgG1, IgG2a, IgG2b or IgG3 antibodies (Southern Biotech, USA) at a concentration of 1:400 in BSA₅PBST for 2 h. Plates were developed with developing solution (hydrogen peroxide, citric acid and ABTS) and incubated for 10-15 min with gentle agitation to observe a colour change. The reaction was stopped with 50 mM sodium fluoride. Plates were read at dual wavelengths of 505 and 595 nm on plate reader (LabSystems Multiskan Multisoft microplate reader). The titers of antibody are expressed as the reciprocal of the highest dilution of serum required to achieve an OD₆₀₀ of 0.2.

Cell-based ELISA was carried out to determine the inhibitory effect. After 48 h of virus infection without or with gallic acid (Sigma-Aldrich Corporation, St. Louis, MO, USA), herb extracts, or Triphala formulation treatment, the infected cells were harvested and stained for intra-cellular viral protein antigen. The infected cells were fixed with 4% paraformaldehyde at room temperature for 15 min and washed twice with ice-cold phosphate-buffered saline (PBS) before permeabilized with 0.2% Triton X-100 (15 min at room temperature). The cells were blocked with 1% bovine serum albumin (BSA) in PBS for 30 min and incubated for at least 3 h with monoclonal anti-DENV E antibody clone 4G2 at 37 °C. Cells were washed three times with PBS containing 0.5% Tween-20 (PBST) and further incubated for 30 min followed by horseradish peroxidase-conjugated rabbit anti-mouse IgG (Dako, CA, USA) (at 1:2000 dilution). Cell plates were washed before adding TMB (3,3,5,5-tetramethylbenzidine) substrate (Invitrogen, Carlsbad, CA, USA), and the absorbance at an optical density (OD) of 650 was measured, wherein the mock control was set as blank. The absorbance was used to calculate the % E antigen compared to that of the non-treatment control (set as 100% E antigen) as the following equation:

This was performed by the measurement of nitrotyrosine formation using Western immunoblot as described previously [22 (link)]. In brief, after tissue preparation (homogenization and centrifugation) the supernatant was collected and the protein concentration was determined by the Lowry method. Following electrophoresis the proteins were transferred onto PVDF membrane. After incubation with a monoclonal anti-nitrotyrosine antibody (MAB5404, Chemicon, USA), and horseradish peroxidase-conjugated rabbit anti-mouse IgG (P0161, Dakocytomation, Denmark) as the secondary antibody, the membrane was developed with an enhanced chemiluminescence kit (ECL Plus, GE Healthcare, UK), exposed to X-ray film and scanned. ImageJ (NIH, Bethesda, MD) was used to determine the density of nitrotyrosine bands. Equal loading was controlled using Coomassie Blue staining.