Rabbit anti mouse igg hrp

Knock out cells were tested for the presence of CD9 and CD81 at the protein level using 15% SDS-PAGE and Western blotting. A549 cells were lysed with 100 µl lysis buffer (25 mM Tris–HCl pH 7.4 containing 150 mM NaCl, 1% sodium deoxycholate, 1% TritonX-100, 0.1% SDS and 1 mM EDTA and 5% glycerol) and 1 µl protease inhibitor (Halt protease and phosphatase inhibitor cocktail (100X) (Thermo Fisher, Waltham, MA, USA) at 4 °C for 30 min. The lysed cells were centrifuged at 15,000 × g at 4 °C for 15 min. The protein concentration of the supernatant was quantified using a BCA kit (Thermo Fisher, Waltham, MA, USA). Cell lysates were diluted with sample buffer and heated at 100 °C for 5 min. Thirty µg of protein was run on 15% SDS gels, transferred to a nitrocellulose membrane and probed with monoclonal anti-CD9 antibody (clone 602.29) or anti-CD81 antibody (clone 1D6, Bio-Rad, USA) at dilution of 1:1000. Rabbit anti-mouse IgG/HRP (Dako Cytomation, Denmark) at dilution of 1:2000 was used as secondary antibody. BM Chemiluminescence Blotting Substrate (Roche, Germany) was added on the membrane before exposure to an X-ray film.

Cell extracts preparation for immunoblotting was performed as described before [43 (link)]. In brief, cells were collected and washed once with cold PBS. The pellets were resuspended in RIPA buffer with fresh prepared EDTA-free protease inhibitor (Roche) and incubated on ice for 15 min and then sonicated. Protein concentration were determined using the Quick Start Broadford Protein Assay (Bio-Rad). 40 μg of total protein extract from each of the samples was run on 10% SDS PAGE gels (Bio-Rad). The following antibodies were used for immunoblot detection, rabbit polyclonal anti-RMI2 (1:1000) (Abcam), mouse monoclonal anti-α-tubulin antibodies (1:1000)(Sigma-Aldrich), swine anti-rabbit IgG-HRP (1:10,000)(Dako) and rabbit anti-mouse IgG-HRP (1:10,000)(Dako). ECL immuno-blotting substrate (Pierce) was used according to the manufacturer’s instructions.

Kidney tissue samples from 30 week-old female IL-17RA KO lpr and B6/lpr mice were frozen in Tissue-Tec O.C.T. Compound (Sakura Finetek Europe B.V) and stored at −80 °C or embedded in formalin. Two µm sections of formalin-fixed, paraffin-embedded kidney tissues were cut and were routinely stained with haematoxylin or eosin (H&E) and periodic acid- Schiff (PAS) for evaluation of kidney pathology. Complement C3 and IgG staining was performed on 5 µm frozen kidney sections with 1 µg/ml rabbit anti-C3 antibody (Thermoscientific) followed by goat-anti-rabbit IgG-HRP (Dako). For IgG staining rabbit anti-mouse IgG-HRP (Dako) was used. Peroxidase activity was detected with DAB and sections were counterstained with Mayer’s hematoxylin. All sections were scored digitally after examination using a Nanozoomer Digital Pathology Scanner (NDP Scan U10074-01, Hamamatsu Photonics K.K., Japan) and quantified ((number of positive pixels* 0.5) + number of strong positive pixels/total pixels) with software of ImageScope Viewer (V11.2.0.780 Aperio, e-Pathology Solution, CA, USA).
HMGB1 and CD3 staining was performed on 2 µm paraffin sections using polyclonal anti-HMGB1 (Abcam, Cambridge, UK) and polyclonal anti-CD3 (Dakocytomation).

Binding of soluble FcαRI or IgA Ab to plate‐bound peptides was tested with Pepscan‐based ELISA's, which were adapted according to the method previously described 19, 43, 44. The samples were washed to remove unbound fragments after each incubation step. The 455‐well credit‐card format polypropylene cards containing the covalently linked IgA‐peptides were incubated with blocking solution (4% horse serum, 5% ovalbumin (w/v) in PBS/1% Tween). Next, peptides were incubated with soluble FcαRI or 293T cells transfected with FcαRI and subsequently with mouse anti‐human FcαRI IgG mAb (1 μg/ml; BD, Franklin Lakes, NJ). Then, peptides were incubated with rabbit anti‐mouse IgG‐HRP (1/1000, Dako, P0212) for 1 h at RT. Alternatively, after blocking, the covalently linked FcαRI‐peptides were incubated with pooled human serum IgA (Cappel™, MP Biomedicals, Santa Ana, CA), and rabbit anti‐human IgA‐HRP (1 μg/mL, Dako, P0212) was added. The peroxidase substrate 2,2′‐azino‐di‐3‐ethylbenzthiazoline sulfonate (ABTS) and 2 μL of 3% H₂O₂ was added (1 h, RT). Colour development was measured, which was quantified with a charge coupled device (CCD)‐camera and an image processing system. Values mostly ranged from 0 to 3000, a log scale similar to 1–3 of a standard 96‐well plate ELISA‐reader.

For western blot analysis cells were washed with Phosphate-buffered saline (PBS) and lysed in Radioimmunoprecipitation assay (RIPA) buffer with complete Mini EDTA-free protease inhibitor tablets (Roche, Welwyn Garden City, UK) and the phosphatase inhibitor cocktail PhosSTOP (Roche, Welwyn Garden City, UK). The protein quantification and western blot was performed as described earlier [16 (link)]. PIAS1 XP^® Rabbit mAb (1:500, D33A7, Cell Signaling, Frankfurt am Main, Germany), Anti-β-Actin monoclonal (1:5000, AC-15, Sigma-Aldrich GmbH; Munich; Germany) and Anti-Lamin A antibody (1:1000, Abcam, Cambridge, UK) were used as primary antibody. Rabbit Anti-Mouse IgG, HRP and Goat anti-rabbit IgG, HRP (both: 1:1000, Dako, Frankfurt, Germany) were used as secondary antibody.

A F-bottom high binding 96-well microtiter plate (Greiner Bio-One GmbH) was coated with 50 μL recNP and recombinant maltose binding protein (MBP, 1 μg/mL) and incubated overnight at 4°C. The plate was washed with water and washing buffer (phosphate-buffered saline/0.5% Tween-20, PBS-T), and 200 μL blocking solution (PBS-T/5% milk powder) was added for 20 min. The blocking solution was discarded, and mAbs were added at 100 pg/mL in 50-μL blocking solution to recNP and MBP and incubated for 1 h. The plate was washed again, and 50 μL rabbit anti-mouse IgG/HRP (Dako) was added at a 1:1000 dilution for another 1 h incubation. The plate was washed, and 50 microliter TMB substrate solution was added. All incubations took place at room temperature. After 5–10 min, the enzymatic reaction was stopped by the addition of 50 μL 1 N H₂SO₄, and light absorption was measured with a photometer at 450 nm using 570 nm as a reference wavelength. Measurements were taken in duplicates. To obtain the final OD, the OD obtained with the control protein MBP was subtracted from the OD obtained with recNP.