Horseradish peroxidase conjugated goat anti mouse igg h l

For the detection of serum anti-NS1 IgG antibodies, an in-house ELISA was performed in 96-well microtiter plates (Thermo Scientific, Cat. No. 3455) coated with 100 ng in 100 µL per well of the YF NS1 protein (The Native Antigen Company, Cat. No. YFV-NS1-500). After incubating overnight, plates were washed (1× PBS, 0.05% tween-20) and blocked for 24 h with a blocking buffer (PBS, 0.05% tween-20, and 1% powdered milk), followed by a wash and incubation for 2 h with eight 5-fold serial dilutions of mouse or macaque serum (1:50 primary dilution) in 100 µL of blocking buffer. After washing, plates were incubated for 1 h with 1:5000 diluted horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) (Invitrogen, Cat. No. 31430) or 1:2000 diluted horseradish peroxidase-conjugated goat anti-human IgG (H+L) (Promega, Cat. No. W4031) in 100 µL of blocking buffer, developed with 1-Step™ Ultra TMB-ELISA substrate solution (Invitrogen, Cat. No. 34028), stopped with 450 nm stop solution for TMB substrate (abcam, Cat. No. ab171529) and the absorbance was measured at 450 nm. The absorbance cutoff value was defined as the mean of a naive serum sample for each plate plus three times the SD. The endpoint titer of a sample was defined as the reciprocal of the highest dilution that gave a positive reaction (above the cutoff).

Brain homogenates (10 or 20 μg in 20 μl) were optionally treated with a final concentration of 25, 12.5 or 6.25μg/ml of trypsin or proteinase-K (37°C), or 1000, 100 or 10 μg/ml of thermolysin (70°C) for 30 min. Samples were subsequently mixed with 4x loading dye and denatured at 95°C for 5 min. Fifteen microliters of each sample were separated on a 4–12% Bis-Tris SDS polyacrylamide gel (NuPAGE, Invitrogen) and electrotransferred to nitrocellulose membranes. Membranes were blocked for 1 hour in 5% Topblock (Fluka) in Tris-buffered saline supplemented with Tween 20 [150 mM NaCl, 10 mM Tris-HCl, 0.05% Tween 20 (v/v)] and incubated overnight with anti-PrP antibodies POM1, POM3, POM5, POM6, POM15 or POM19 (200 ng/ml). As secondary antibody, horseradish peroxidase-conjugated goat anti mouse IgG (H+L) (1:10.000, Invitrogen) was used and immunoreactivity was visualized using chemiluminescence (Luminata crescendo, Merck Millipore).

The mBMSC culture and differentiation medium was purchased from Cyagen (China). The chondrocyte activator IL-1β was purchased from Sigma-Aldrich (USA). The primary antibodies used in this study included mouse anti-CD9, CD63, CD81, and SOX9 (Santa Cruz Biotechnology, USA); rabbit anti-COL II (Santa Cruze Biotechnology); rabbit anti-PDGFBB, VEGF, SDF-1, COL X, anti-cleaved caspase-3, Bcl-2, Bax, and GAPDH (Abcam, USA); rabbit anti-TGFβ1, Smad2/3, p-Smad2/3, EKR1/2, p-EKR1/2, p38, and p-p38 (Cell Signaling Technology, USA); rabbit anti- COL X and Ki67 (Novus, USA); rabbit anti-Aggrecan, and MMP13 (Proteintech; China).The secondary antibodies used in this study included Alexa-488 conjugated-goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch, USA); horseradishperoxidase–conjugated-goat anti-rabbit IgG (H + L) and horseradish peroxidase–conjugated-goat anti-Mouse IgG (H + L) (Invitrogen, USA); Nuclei was stained with DAPI dihydrochloride (Thermo Fisher Scientific, USA). Flow cytometry anlysis was performed to identify the characterization of mBMSCs stained with FITC-conjugated or PE-conjugated anti-mouse CD44, CD45, CD90, and CD105 (BD, USA).