Hrp conjugated goat anti mouse secondary antibody

The 96-well plates were coated with YG8–1 overnight. After blocking with 5% non-fat milk at 37 °C for 1 h, serially dilutions of YG8–2 and YG8–3 were added to the wells respectively, which mixed with 10 μg/mL GST-HlgB, after incubated at 37 °C for 1 h and washed 5 times with PBST, the monoclonal mouse anti-GST antibodies (Transgene, HT601, 1:1000) were added for 45 min at 37 °C. Followed by the addition of a HRP-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, 115–035-174, 1:20000) for 30 min at 37 °C. The plates were washed 3 times and incubated for 30 min with TMB. Optical density at 450 nm was measured in a microtiter plate reader.

For western blot analysis, we used an anti-SMN mouse monoclonal antibody (BD Transd Lab, clone 8, #610646; 1:10,000), an anti-Tubulin mouse monoclonal antibody (Sigma, clone DM1A, #T9026; 1:50,000), and a HRP conjugated goat anti-mouse secondary antibody (Jackson #115-035-044; 1:10,000). For spinal cord immunohistochemistry, we used goat anti-ChAT (Millipore #AB144P; 1:100), guinea pig anti-VGluT1 (Covance, custom made; 1:5,000) [18 (link)], rabbit anti-p53 (Leica Novocastra #NCL-p53-CM5p; 1:1,000) and rabbit anti-phosporylated-p53^S15 (Cell Signaling #9284, Lot: #15; 1:250) as primary antibodies. For muscle immunohistochemistry, we used guinea pig anti-Synaptophysin 1 (Synaptic Systems #101–004; 1:500), rabbit anti-Neurofilament M (Millipore #AB1987; 1:500), and Alexa Fluor™ 555 conjugated α-bungarotoxin (Invitrogen, #B35451; 1:500). Species-specific secondary antibodies coupled to Cy3 or Cy5 were used as appropriate (Jackson ImmunoResearch Laboratories, Inc; 1:250).

Meningococcal capsular group Y polysaccharide-specific IgG antibodies were measured by ELISA following a previously described method [40 (link)]. Briefly, immulon-2 microtiter plates (Thermo Fischer Scientific, MA, USA) were coated with capsular group Y meningococcal polysaccharide (5 μg/ml) (NIBSC, England) conjugated to 5 μg/ml of methylated human albumin (NIBSC, England) in sterile PBS. Following blocking, two-fold dilutions of test sera were assayed in duplicate. After overnight incubation at 4°C, microtiter plates were developed with HRP-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Inc. PA, USA) at a 1:10,000 dilution for 2.5 h at room temperature, followed by the chromogenic substrate tetramethylbenzidine (Sigma–Aldrich, England). The reaction was stopped after 30 min with 2 M H₂SO₄. The optical density of each well was read at 450 nm. Endpoint-titres were determined as the reciprocal of the dilution giving an OD450 nm reading above that obtained for naive control wells plus 2 x SD of 6 replicates for each plate.

Cell samples were harvested using NP-40 cell lysis buffer 24 h post-PRRSV inoculation unless noted otherwise. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) and samples were mixed with 5× SDS sample loading buffer before samples containing equal amounts of total protein were loaded onto 12% SDS-PAGE gels and separated proteins were transferred onto PVDF membranes as described previously [36 (link)]. Membranes were probed with Mab2-5G2 or 6D10 (against the PRRSV-N protein) or anti-β-actin mAb (Abcam, Cambridge, MA, USA). Specific binding of antibodies to targets was detected using horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) and visualized using ECL substrate (Beyotime, Beijing, China).

The breast tissue microarrays were purchased from Novus Biological (Littleton, CO). TMA included 40 breast cancer infiltrating ductal carcinoma, 10 metastatic lymph node and 9 adjacent normal breast tissues. Clinico-histopathologic characteristics of the subjects in the tissue microarray study included grade, age, hormone status and clinical stage, according to information provided by the suppliers. Tissues were de-paraffinized in xylene, rehydrated in graded alcohols and endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 5 min. Slides were treated with LGLRGSL phage (10¹⁰ pfu) overnight. Slides were subsequently washed and blocked by 3% goat serum at RT for 1 h in humidity chambers. Slides were then treated with M13-pIII phage monoclonal antibody (NEB, MA) or Vimentin antibody (Cell Siganling, Danvers, MA, United States) (1:100) and then subsequently with HRP conjugated goat anti-mouse secondary antibody (Jackson Immunoresearch Laboratories Inc., West Grove, PA, United States) for 40 min. The antigen-antibody reaction was visualized after applying diaminobenzidine (Sigma-Aldrich, MO, United States) for 7 min. The slides were counterstained with hematoxylin (Sigma-Aldrich, MO, United States) for 1 min. Slides were dehydrated in alcohols and cleared in xylene baths before being mounted with Permount media.

Samples were separated by 10% SDS-PAGE gel (Bio-Rad Laboratories, Inc., Hercules, CA), and transferred to nitrocellulose membrane. Membranes were blocked with 5% skim milk in 1x PBST (1xPBS, 0.075% Tween20) at 4°C o/n, then incubated with a mouse monoclonal Factor B antibody specific for the Bb subunit (1:200 in 5% BSA, 1xPBST; Santa Cruz Biotechnology, Inc., Dallas, TX; F-7: sc-271636) at 4°C o/n. Membranes were washed x3 with 1xPBST, followed by a 2-hour incubation at room temperature with polyclonal HRP-conjugated goat anti-mouse secondary antibody (1:4000, Jackson ImmunoResearch Inc, West Grove, PA, 115-035-062, RRID: AB_2338504, in 5% BSA, 1xPBST). Membranes were washed x5 in 1xPBST, then incubated with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, Rockford, IL) for 1 min. Protein bands were visualized using Classic X-ray Film (Research Products International, Mt. Prospect, IL).

Paraffin-embedded human kidney tissue sections (4 µm thick, from four different donors) were deparaffinized, rehydrated, and subjected to microwave-based antigen retrieval in citrate buffer. Endogenous peroxidases were blocked with 0.3% (v/v) H₂O₂ in PBS for 10 min. Non-specific immunoglobulin binding sites were blocked with normal goat serum. Sections were subsequently incubated with mouse anti-human C5aR1 primary antibody (P12/1, AbD Serotec) (1:300 dilution) at 4°C overnight. Non-specific mouse IgG2a served as a negative control. Sections were then incubated for 1 h with HRP-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Lab Inc., 1:1,000 dilution) at room temperature. The antibody staining was visualized by DAB solution according to manufacturer’s instructions. The stained sections were imaged with automatic slide scanner (Axio Scan Z1, Zeiss, Germany).