Anti mouse igg hrp

Whole sperm protein was extracted as previously described (66 (link), 67 (link), 68 ). In short, mouse epididymal spermatozoa (1 × 10⁶ cells) washed in PBS were directly lysed in 20 μl 2× SDS sample buffer or 8 M urea. The whole sperm lysate was centrifuged at 15,000g, 4 °C for 10 min. Supernatant were adjusted to 50 mM DTT and denatured at 95 °C for 10 min before loading to a SDS-PAGE gel. The proteins were transferred on a nitrocellulose membrane. The membrane was incubated overnight at 4 °C with primary antibody, followed by washing with PBST and incubation with appropriate secondary antibody for 1 h. Bands were visualized using chemiluminescence and imaged by ChemiDoc (Bio-Rad). Primary antibodies used for Western blotting were rabbit anti-mouse TXNRD3 (1:2000), GPX4 (1:2000), ANT4 (1 μg/ml), P2X2 (1 μg/ml), TOM20 (0.2 μg/ml), TXNRD1 (1 μg/ml), TXNRD2 (1 μg/ml), and mouse anti-acetylated tubulin (1: 20,000). Secondary antibodies were anti-rabbit IgG-HRP (1:10,000) and anti-mouse IgG-HRP (1:10,000) from Jackson ImmunoResearch. Normalized expression was measured by ImageJ software (v1.53).

MaxiSorp plates (Thermo Scientific) were coated with lysate, diluted 1:1,000 in PBS, from RVFV-infected Vero-E6 cells or with lysate from uninfected Vero-E6 cells to act as a negative control (McElroy et al., 2009 (link)). Plates were left at 4°C overnight, and then blocked in blocking buffer (5% non-fat milk in PBS-0.1% Tween 20) at 37°C for 1 h. Terminal mouse serum samples were serially diluted in blocking buffer, and then incubated on blocked plates at 37°C for 2 h. All serum samples were assayed in duplicate alongside normal mouse serum as a negative control. After incubation with sera, plates were washed three times with PBS-0.1% Tween 20 (PBST), and then incubated for 1 h at 37°C in anti-mouse IgG-HRP (Jackson ImmunoResearch) diluted 1:5,000 in blocking buffer. Following three PBST washes, the plates were incubated in tetramethylbenzidine (TMB) substrate, and then stopped with TMB stop solution. Plates were read at 450 nm and the raw data were analyzed in Excel by subtracting the negative control absolute values from those of the RVFV lysate plate. The endpoint ELISA titer for each mouse was defined as the highest dilution of serum that resulted in an OD value at least two standard deviations above the average obtained from all negative mouse serum control wells.

Virus titer from each A (H7N9) passage was determined by the TCID50 assay. To prepare for the assay, 3 × 10⁴ MDCK cells/well were seeded in the 96-well plate. After 24 hours, the virus supernatants collected from hHACs were serially diluted at 1:10. MDCK cells were infected by the diluted supernatants at aliquots of 50 μl and incubated at 37 °C 5% CO₂ for 1 hour. The infected cells were washed with PBS and cultured in MEM with 2% FBS and 1 μg/ml TPCK-trypsin. Cells were later incubated in a 37 °C, 5% CO₂ incubator for 24 hours and then fixed in 80% acetone for 10 min. Fixed cells were washed twice with wash buffer and incubated with anti NP monoclonal antibody (1:1500, Santacruz) at 37 °C for 1 hour. Cells were washed with wash buffer 3 times and incubated with anti-mouse IgG-HRP (1:2000, Jackson) at 37 °C for 1 hour. Cells were washed with wash buffer 6 times, reacted with OPD substrate, and stopped by 1N of sulfuric acid. OD values at 492nm were read by EZRead 400 microplate (biochrom). TCID50 values were calculated as per standard protocol.

Established protocols were followed for SDS-PAGE/immunoblotting [53 (link)]. Equal amounts of total proteins for each sample (10–20 μg) were separated on SDS-PAGE and then electrotransferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked with 5% nonfat milk for 1 h and probed with specific primary antibodies overnight at 4 °C followed by incubation with goat anti-rabbit IgG-HRP (1:15,000; 111-035-144) or anti-mouse IgG–HRP (1:15,000; 115-035-003) or donkey anti-goat IgG-HRP (1:15,000; 705-035-003) (Jackson ImmunoResearch Laboratories; West Grove, PA) at room temperature for 1 h. To control for false-positive signals, we used rabbit normal IgG (011-000-003, Jackson ImmunoResearch Laboratories) and an unrelated goat IgG (cathepsin D, sc-6486, Santa Cruz Biotechnology) at comparable working concentrations. All blots were developed using the BioRad Clarity Western ECL substrate and captured using ChemiDoc XRS + (Bio-Rad); only blots within signals in the linear range were quantitated using the ImageLab 3.0.1 software (Bio-Rad).

Plates were coated overnight at 4°C with 1.0 μg/mL anti-CD19 antibody FMC63 (#NBP2-52716, Novus Biologics, Centennial, CO) or 1.0 μg/mL purified Her2-Fc (#HE2-H5253, AcroBiosytems, Newark, DE), in 0.1 M carbonate, pH 9.5. The plate was blocked with 0.3% non-fat milk in TBS for 1 hour at room temperature. After washing in TBST 3 times, the bridging protein was titrated from a starting concentration of 5 μg/mL using serial 3-fold dilutions in TBS/1% BSA and incubated 1 hour at room temperature. Detection with Her2-Fc, biotinylated Her2 (#He2-H822R, AcroBiosystems), or antibody FMC63 was performed by adding the detection reagent at 1.0 μg/mL and incubating for 1 hour at room temperature. The plates were then washed 3 times, followed by a 1 hour incubation with a 1:2000 dilution of anti-human IgG-HRP (#109-035-088, Jackson ImmunoResearch, West Grove, PA) for Her2-Fc detection, HRP-steptavidin (#21130, Thermo Fisher) for biotinylated Her2 detection, and anti-mouse IgG-HRP (#115-035-062, Jackson ImmunoResearch) for FMC63 detection. Then, 1-Step Ultra TMB-ELISA solution (#34028, Thermo Fisher) was added to develop the peroxidase signal, and the plate was read at 405 nm. Curves were fit using a four parameter logistic regression to calculate the EC₅₀.