Horseradish peroxidase conjugated goat anti mouse igg

MDCK cells were seeded in a 96-well plate (1 × 10⁴ cells/well). (+)-(S)-Bakuchiol, (−)-(R)-bakuchiol, or ME was mixed at a concentration of 12.5–50 μm with influenza A virus (A/PR/8/34, A/CA/7/09, or A/Aichi/2/68) at an MOI of 0.1 in the infection medium and incubated for 30 min at 37 °C under 5% CO₂. DMSO (0.125–0.5%) was used as the negative control. Each mixture was added to the cells and incubated for 24 h at 37 °C under 5% CO₂. The cells were then fixed with 4% paraformaldehyde in PBS for 30 min at 4 °C before permeabilization with 0.3% Triton X-100 for 20 min at room temperature. Mouse antibodies detecting the NP of A/PR/8/34 and A/Aichi/2/68 (FluA-NP 4F1, SouthernBiotech) or the NP of A/CA/7/09 (AA5H, AbD Serotec) were used as primary antibodies, as appropriate (26 (link)). Horseradish peroxidase-conjugated goat anti-mouse IgG (SouthernBiotech) was used as the secondary antibody. To visualize the infected cells, TrueBlue peroxidase substrate (KPL) was added and incubated for 15 min; color development was terminated by washing with H₂O. The wells were photographed under a microscope, and the stained cells were counted. Each half-maximal (50%) inhibitory concentration (IC₅₀) value was then calculated based on the cell numbers.

Tumor or thymus tissues collected from tumor-bearing mice treated with PBS, RdB, RdB/shVEGF, RdB/IL12, or RdB/IL12/shVEGF were fixed in 10% neutral buffered formalin (Junsei Chemical, Japan), embedded in paraffin, and cut into 4-μm-thick sections. Representative sections were stained with H & E, and then examined by light microscopy. To detect lymphocytes and DCs, tumor tissues were frozen in Optimal Cutting Temperature compound (Sakura Finetec, Torrance, CA, USA) and cut into 10-μm-thick sections. The cryosections were incubated with rat anti-mouse CD4 monoclonal Ab (BD Biosciences Pharmingen), rat anti-mouse CD8 monoclonal Ab (BD Biosciences Pharmingen), or mouse anti-mouse PCNA monoclonal Ab (DAKO, Denmark) as the primary Ab, and then treated with horseradish peroxidase-conjugated goat anti-rat IgG (BD Biosciences Pharmingen) or horseradish peroxidase-conjugated goat anti-mouse IgG (Southern Biotech, Birmingham, AL, USA) as the secondary Ab. Diaminobenzidine/hydrogen peroxidase (DAKO) was used as the chromogen substrate. All slides were counterstained with Meyer's hematoxylin (Sigma). The expression levels of CD4, CD8, and PCNA were semi-quantitatively analyzed using MetaMorph^® image analysis software (Universal Image Corp., UK). Results are expressed as the mean optical density of five different digital images.

The cells were lysed in a buffer containing 125 mm Tris-HCl, pH 6.8, 5% SDS, 25% glycerol, 0.1% bromphenol blue, and 10% β-mercaptoethanol and boiled for 5 min. The cell lysates were then separated on a 10% polyacrylamide gel. The proteins were transferred to a polyvinylidene fluoride microporous membrane (Millipore). FluA-NP 4F1 (SouthernBiotech), a goat anti-influenza A viral NS1 antibody (vC-20, Santa Cruz Biotechnology, Inc.), a rabbit anti-firefly luciferase polyclonal antibody (MBL, Nagoya, Japan), and a rabbit anti-Renilla luciferase polyclonal antibody (MBL) were used as primary antibodies to detect their respective proteins. A rabbit anti-β-actin antibody (13E5, Cell Signaling) was used as an internal control. The secondary antibodies, horseradish peroxidase-conjugated goat anti-mouse IgG (SouthernBiotech), donkey anti-goat IgG (sc-2020, Santa Cruz Biotechnology), or goat anti-rabbit IgG (KPL), were used as appropriate. The signals were detected using Western Lightning ECL Pro (PerkinElmer Life Sciences). Signal intensities were measured using ImageJ software, and the protein levels of firefly and Renilla luciferase were normalized to that of β-actin.

Two groups each of 6 mice received intraperitoneal administration of either commercial L-asparaginase (250 U/kg) or Streptomyces brollosae NEAE-115 L-asparaginase (250 U/kg) twice a week for 4 weeks. Levels of specific antibodies (IgG immunoglobulin) against L-asparaginase or commercial product were measured in sera using ELISA method and microplate reader was used at absorbance of 450 nm. The direct enzyme-linked immunosorbent assay (ELISA) was carried out to evaluate the presence of asparaginase–specific IgG antibodies in serum samples by employing horseradish peroxidase–conjugated goat-anti mouse IgG (from Southern Biotech). All ELISA steps were conducted at room temperature, and 2.5% casein (w/v) was utilized as binding and blocking buffer. The assay steps and conditions were as follows: 2 h blocking of asparaginase-coated plates followed by extensive washing using PBS-Tween, incubated with diluted serum samples for 2 h, washed, incubated with detection antibody for 1 h, washed, developed with 3, 3′, 5, 5′-tetramethylbenzidine substrate for 30 min, and quenched with 9.8% (v/v) H₂SO₄ in water. IgG titers were calculated.

For the detection of anti-peptide antibodies, high-binding 96-well flat-bottom plates Nunc MaxiSorp (Thermo Fisher Scientific) were coated overnight with 5 μg mL^-1 of each peptide in PBS. The next day, plates were blocked with 1% bovine serum albumin (BSA) in PBS, for 1 h at 37°C and washed with 0.02% (v/v) Tween-20 in PBS. Single dilutions of the sera were performed in PBS 0.25% BSA and incubated, in duplicate, for 1h at 37°C followed by incubation with a horseradish peroxidase-conjugated goat anti-mouse IgG (1:5,000, Southern Biotech). Plates were washed and revealed using OPD (Sigma), according to the manufacturer’s instructions. The reaction was stopped with 3M HCl and the plates read at 492nm in a Synergy 2 microplate reader (Biotek Instruments) with the Gen5 software (Biotek Instruments).

Cell extracts for western blot analysis were prepared in ONYX lysis buffer (20 mM Tris-HCl pH 7.4, 135 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 1% Triton X-100, 10% glycerol). Antibodies used include: rat anti-BIM (clones 3C5 and CF7, ENZO Life Sciences,^{29 (link)} Waterloo, NSW, Australia), mouse anti-β-actin (Sigma AC-40, Rowville, VIC, Australia). Horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rat IgG antibodies (both from Southern Biotech, Birmingham, AL, USA) served as secondary reagents and the enhanced chemoluminescence (ECL; GE Healthcare, Rydalmere, NSW, Australia) system was used for detection. WBC analysis was performed with an ADVIA blood analyzer (Siemens Healthcare Diagnostics, Tarrytown, NY, USA).