Hrp conjugated goat anti rabbit antibody

Rat spinal neurons were identified by immunocytochemistry staining. Briefly, at 7 d in vitro, cultures on PLL-coated coverslips were fixed with 4% paraformaldehyde (PFA) for 30 min, and rinsed three times with 0.01 M phosphate-buffered saline (PBS) and blocked with 5% BSA for 20 min at RT. The monoclonal rabbit anti-rat primary antibody against β-tubulin III (Sigma) was added to the cell slides and co-cultured with the cells in a humidified chamber overnight at 4°C. After three washes with PBS, cultures were incubated for 1 h at 37°C with the HRP-conjugated goat anti-rabbit antibody (Sigma). Then cultures were stained with peroxidase substrate diaminobenzidine. 4′, 6-diamidino-2-phenylindole (DAPI; Sigma) was used to stain nuclei of all cells, including neurons and glial cells. The purity of cultured spinal neuron was expressed as the percentage of β-tubulin III positive cells relative to the total number of DAPI labeled nuclei.

SepSecS mutant enzymes were resolved either on SDS- or native PA gels and transferred onto Low Fluorescence 0.2 μm PVDF membrane (GE Healthcare). For SDS-PAGE blots either 25 or 10 ng of total protein was loaded onto the gel for probing for either the 6xHis-tag or GroEL, respectively. For native PAGE blots, either 3 μg or 350 ng of total protein was loaded for probing for the 6xHis-tag or GroEL, respectively. The membrane was blocked in blocking buffer (5% nonfat dry milk in PBST, 50 mM phosphate buffer, pH 7.6, 150 mM NaCl and 0.1% Tween-20) for 1 h at room temperature. Subsequently, the membrane was incubated with either an anti-His-tag antibody conjugated with horseradish peroxidase (R&D Systems) or anti-GroEL (Sigma-Aldrich) in blocking buffer overnight at 4 °C. Following primary antibody incubation, the GroEL blot was incubated with an HRP-conjugated goat anti-rabbit antibody (Sigma-Aldrich). Immunoblots were developed by enhanced chemiluminescence using ECL Prime Western Detection Reagent (GE Healthcare) and a Konica Minolta SRX-101A imager with Amersham Hyperfilm ECL (GE Healthcare).

The virulence of S. suis strains 11313 and 11LB5 was evaluated in mouse studies. Briefly, 40 6-week-old female CD1 mice (11 mice/infected group, 7 mice/control group) were injected intraperitoneally (i.p.) with 0.25 mL containing S. suis strain 700794 (positive control), strain 11313 or 11LB5 (5 × 10⁷ CFU total), or sterile THB (negative control). Infected mice were monitored carefully for clinical symptoms and survival time for 10 days. Three mice from each group were euthanized humanely at 3 days post-inoculation (3 dpi), blood and tissue samples were collected. Then, 5 μL of blood or 10 mg tissue sample was plated and cultured on THB agar plates for 24 h. Tissue samples of mice were fixed in 10% formalin buffer and embedded in paraffin, cut in 2–3 µm thick slices and stained by hematoxylin and eosin (H&E), then observed by DM4000B upright fluorescence microscope (Leica, Frankfurt, Germany). To determine the type of glial cells that increased in the brain after infection, immunohistochemistry (IHC) was performed after deparaffinization and antigen retrieval (high pressure, 121 °C, 15 min), by addition of rabbit anti-mouse GFAP or Iba1 antibody to stain astrocytes or microgila (abcam, Cambridge, UK) and HRP-conjugated goat anti-rabbit antibody (Sigma, Saint Louis, MO, USA). Then, DAB (3, 3′-diaminobenzidine) was used to visualize the secondary antibody.

The Sf9 cells were infected with the recombinant baculovirus vAc-prME or the control baculovirus vAc-hsp70-egfp at a multiplicity of infection (MOI) of 5, and the lysates were collected at 72 h post infection (h.p.i.) and prepared for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with Tris-buffered saline (TBS) containing 5% nonfat milk, the membranes were incubated with anti-E and anti-prM pAb as primary antibodies, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (Sigma, St. Louis, MO, USA) as the secondary antibody. Protein band signals were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA).

Cell proteins were extracted using RIPA Lysis Buffer (Beyotime, Shanghai, China), and a qualification analysis was conducted through BCA protein kit (Beyotime). The equal amounts of proteins (20 μg) were subjected to 12% SDS‐PAGE and then transferred onto PVDF membranes (Invitrogen). Electrochemiluminescent (ECL) Detection System (Thermo Fisher Scientific, Waltham, MA, USA) was used for signal detection. Specific proteins were incubated with primary antibody rabbit‐anti‐human BCL2L2 (1:1000, Cell Signaling, Danvers, MA, USA). Anti‐β‐actin (1:500, Sigma‐Aldrich) was used as internal reference, and HRP‐conjugated goat‐anti‐rabbit antibody (1:1000) was used as secondary antibody.

786-0 and ACHN cells were washed with PBS buffer twice and then homogenized in 200μl radioimmuno-precipitation assay (RIPA) buffer containing the protease inhibitors cocktail(1 mmol/L) and phenylmethylsulfonyl fluoride (100μg/mL). Homogenates were centrifuged and supernatants were collected. A total of 50 μg of protein separated by 10% sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membrane were saturated with 5% skim milk in TBST (50 mM Tris–HCl, 150 mM NaCl, 0.1% Tween-20) for 2h and then incubated with primary antibodies at 4°C overnight. The primary antibodies used in this study included rabbit polyclonal antibodies to UTX (1:1,000, Abcam, Hong Kong, China), JMJD3 (1:1500, Abcam), EZH2 (1:500, Santa Cruz Biotechnology, Hong Kong, China), H3K27me3 (1:1,500, Epigentek, Brooklyn, USA), H3 (1:2,000, Sigma-Aldrich, St Louis, USA) and actin (1:2,500, Sigma, St Louis, USA). The membranes were incubated with HRP-conjugated goat anti-rabbit antibody (1:5,000, Sigma) for 1 h at room temperature and then exposed to enhanced chemiluminescence substrate (Millipore, Rockford, USA), and detection was performed using a film.

Determining the exosome content by P-ELISA was accomplished by initially adding 20 μL analyte to the paper-based immunoaffinity devices and incubating for 1 h. Subsequently, the devices were treated with 5 μL of rabbit anti-human anti-CD9 antibody (1 μg/ml in PBS from Sigma-Aldrich, United States ) and incubated for 1 min before being washed 3 times with 20 µL PBS. A similar procedure of adding-incubating-washing was duplicated on the device with 5 µL of HRP-conjugated goat anti-rabbit antibody (Sigma-Aldrich, United States ). Finally, 5 μL of mixed 3,3′,5,5′-tetramethylbenzidine (TMB) (Sigma-Aldrich, United States ) and hydrogen peroxide (Sigma-Aldrich, United States ) (1:1 mix per volume) was added, and the device was incubated for color development (Supplementary Figure S3). The P-ELISA results were photographed with a cell phone camera (iPhone XR) in 8-bit format, and the color intensity from the last reaction step was quantified by ImageJ software.