Hrp conjugated rabbit anti mouse igg

For determination of β-catenin protein expression, deparaffinized tumour sections were incubated with anti-β-catenin IgG (1:25 in 1% v/v goat serum, 1% v/v human serum) overnight and subsequent with HRP-conjugated rabbit antimouse IgG (1:1000; DAKO, Glostrup, Denmark). Staining was performed with 0.5 mg/ml 3,3′-diaminobenzidine in 30 mM imidazole containing 0.03% v/v H₂O₂ and 1 mM EDTA, pH 7.0. As a negative control, the primary antibodies were omitted. For each sample, expression was quantified by measuring the average staining intensity on a scale of 256 channels using ImageJ software in four different random selected regions within the tumour (using haematoxylin and eosin-stained sections). Differences between the groups were tested for significance using the Kruskal–Wallis test. All procedures were performed blind.

Cells were lysed in urea lysis buffer (7 M urea, 0.1 M DTT, 0.05% Triton X-100, 25 mM NaCl, 20 mM Hepes pH 7.5). Equal amounts of protein were separated by SDS polyacrylamide gel electrophoresis (SDS PAGE), transferred to Hybond-C nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK) and hybridised to an appropriate primary antibody and HRP-conjugated secondary antibody for subsequent detection by ECL. Primary antibody against β-actin was purchased from Abcam (Cambridge, UK). Antibodies against Bax and Bcl2 were purchased from Santa Cruz Biotechnology (CA, USA). The secondary antibodies, HRP-conjugated rabbit anti-mouse IgG and swine anti-rabbit IgG, were obtained from DakoCytomation (Glostrup, Denmark).

Cells were lysed in urea lysis buffer (7 M urea, 0.1 M DTT, 0.05% Triton X-100, 25 mM NaCl, 20 mM Hepes pH 7.5). Equal amounts of protein were separated by SDS polyacrylamide gel electrophoresis (SDS PAGE), transferred to Hybond-C nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK) and hybridised to an appropriate primary antibody and HRP-conjugated secondary antibody for subsequent detection by ECL. Antibodies against Fibronectin and β-catenin were purchased from BD Biosciences (Oxford, UK). Anti-Zeb1, Anti-vimentin and β-actin were purchased from Abcam (Cambridge, UK). Anti-COX-2 (C-20) was purchased from Santa Cruz biotechnology (Texas, USA). Secondary antibodies were HRP-conjugated rabbit anti-mouse IgG and swine anti-rabbit IgG, were obtained from DakoCytomation (Glostrup, Denmark).

IgG, IgM, and IgA serum levels were measured using commercially available ELISA kits (Bethyl Laboratories, Nordic BioSite). Levels below the assay range were set to a level that was the lowest detectable level according to the kit.
As a marker for bone resorption, serum levels of C‐terminal type I collagen fragments (CTX‐I) were determined with a commercially available ELISA Crosslaps kit (Immunodiagnostic Systems, Boldon, UK). As a marker of bone formation, serum levels of N‐terminal propeptide of type I procollagen (P1NP) were assessed with a commercially available EIA P1NP kit (Immunodiagnostic Systems).
Levels of mBSA‐specific antibodies were assessed in serum of AIA mice using an in‐house ELISA, as described.⁽³³⁾ Briefly, ELISA plates were coated with 0.01 mg/mL mBSA, blocked and washed with casein (Sigma‐Aldrich) before adding the serum. Bound anti‐mBSA antibodies were detected by horseradish peroxidase (HRP)‐conjugated rabbit anti‐mouse IgG (DAKO, Agilent, Santa Clara, CA USA).

The VRPs were titrated in SK-6(E^rns) cells by end-point dilution and immunoperoxidase staining using the anti-E2 monoclonal antibody (mAb) HC/TC26 [34 (link)] kindly provided by I. Greiser-Wilke (Hannover Veterinary School, Hannover, Germany) and a horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (Dako). Alternatively, the VRPs were titrated in PK-15 cells using the mAb WH211 (APHA, RAE0242) and an HRP-conjugated polyclonal goat anti-mouse serum (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Titration of the PRRSV-2 strain used for challenge was performed in MARC-145 cells using the monoclonal antibody, SDOW17-A (RTI LLC) and HRP-conjugated rabbit anti-mouse serum (Dako). The SK-6(E^rns) cells were maintained as described above, and the PK-15 and MARC-145 cells were cultured in Dulbecco’s modified Eagle medium supplemented with 5% fetal bovine serum (FBS).

Twenty μg of protein extract were denatured, separated by a 4-15% SDS-PAGE (Criterion TGX, Bio-Rad) and transferred to nitrocellulose membrane (Whatman). After blocking the membrane in 5% non-fat milk powder dissolved in PBS with 0.1% Tween, membranes were incubated in 0.5% non-fat milk powder at 4°C overnight with 0.1 μg/mL final concentration of detection antibody (BAF960), 0.1 μg/mL final concentration of capture antibody (MAB9601) or 0.1 μg/mL final concentration of mouse anti-human EpCAM (clone C-10, SCBT). Afterwards, membranes were incubated with a HRP-conjugated rabbit anti-mouse IgG (Dako Cytomation) for capture antibody and a HRP-conjugated rabbit anti-goat IgG (Dako Cytomation) for detection antibody, respectively. Next, a dilution of 1:1,000 for 1 hour at room temperature was prepared. After washing, a chemoluminescent substrate (LumiGLO Reagent and Peroxide, Cell Signaling Technology) was added to the membranes and protein was detected in the Chemidoc XRS station (Biorad Laboratories).
For Dot Blot analysis 10 ng of FLAG-tagged EpCAM produced in HEK293FT cells dissolved in assay buffer (1%BSA/PBS) or serum (n = 6, healthy probands) or ascites (n = 12) were spotted onto nitrocellulose membrane.

All chemicals including cell culture medium (Dulbecco´s modified Eagle´s medium (DMEM)) as well as the catalase-polyethylene glycol (PEG-catalase) were obtained from Sigma or Merck (Darmstadt, Germany) unless otherwise stated. The fetal bovine serum was from Pan Biotech (Aidenbach, Germany). The Protein Assay Kit (Bio-Rad DC, detergent compatible) was from Bio-Rad Laboratories (Feldkirchen, Germany). The enhanced chemiluminescence system (SuperSignal West Pico/Femto Maximum Sensitivity Substrate) was supplied by Pierce (Fisher Scientific, Schwerte, Germany). Penicilin/Streptomycin was obtained from Biochrom (Berlin, Germany) and Glutamax from Gibco (Darmstadt, Germany). MitoTEMPO was purchased from Enzo Life Sciences (Lausen, Schweiz). MitoSOX Red and MitoTRACKER Green FM were obtained from Fisher Scientific (Schwerte, Germany). Beta-actin antibody was purchased from Cell Signaling (Massachusetts, USA). The polyclonal rabbit α-hapten antibody directed against dimedone tagged sulfenic acids was kindly provided from K.S. Carroll [35 (link)]. TOM-20 antibody was purchased from Santa Cruz Biotechnology (Dallas, USA). DMSO was obtained from Roth (Karlsruhe, Germany). Horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG from Dianova (Hamburg, Germany) and HRP conjugated rabbit anti-mouse IgG from Dako (Glostrup, Denmark) were used as a secondary antibody.