Hrp conjugated anti mouse igg antibody

Cell lysates were loaded on NuPAGE 10% Bis–Tris Gel (ThermoFisher Scientific) for electrophoresis and later transferred to a nitrocellulose membrane. The membrane was blocked with skimmed milk 6% for 1 h and incubated with the antibodies. The primary antibodies used were rabbit polyclonal anti-Bcl6 (ThermoFisher Scientific, Ref PA5-27390, dilution 1:1000) and mouse anti-ß-actin (Sigma-Aldrich, clone AC-74, dilution 1:2500). The secondary antibodies were goat anti-rabbit horseradish peroxidase- (HRP-) conjugated antibody (ThermoFisher Scientific, reference 31460) and an anti-mouse IgG HRP-conjugated antibody (Abcam, reference ab6789). The membranes were revealed using an ECL system (Cytiva, Marlborough, MA) using a ChemiDoc XRS+ (Bio-Rad, Hercules, CA USA), and the obtained picture was analyzed using the ImageJ software (NIH).

The purified EV fractions were mixed with 4 × SDS sample buffer (0.2 M Tris–HCl, pH 6.8; 8% SDS; 40% glycerol; 0.4% bromophenol blue) and incubated at 37 °C for 30 min prior to electrophoresis (for CD9 and CD63 detection). The purified EV fractions were mixed with 4 × SDS sample buffer containing 2-mercaptoethanol and incubated at 65 °C for 5 min prior to electrophoresis (for TSG101 and Calnexin detection). EVs bound to the K8-peptide magnetic beads were eluted and lysed with 1 × SDS sample buffer (50 mM Tris–HCl, pH 6.8; 2% SDS; 10% glycerol; 0.1% bromophenol blue). MCF7 cells were disrupted by ultrasonication (Branson, Danbury, CT, USA) and used as a cell lysate. These samples were subjected to SDS–polyacrylamide gel electrophoresis (PAGE) and immunoblotting was performed using an anti-human CD9 antibody (1:2000, clone 12A12, Cosmo Bio Co., Ltd), an anti-human CD63 antibody (1:2000, clone 3–13, Wako), an anti-human TSG101 antibody (1:1000, ab30871, Abcam), or an anti-human Calnexin antibody (1:2000, ab22595, Abcam). HRP-conjugated anti-mouse IgG antibody (1:10,000, Abcam) and HRP-conjugated anti-rabbit IgG antibody (1:10,000, Abcam) were used as the secondary antibodies as appropriate. Immunoreactive proteins were detected and analyzed using a EzWestBlue substrate (ATTO, Tokyo, Japan).

Purified 100-fold diluted engineered exosomes (C9HuH-7exo, LC9-293exo and HN3C9-293exo) were used to analyze the size distribution by DLS (Zetasizer Nano ZS, Malven Instruments, UK) method. TEM (JEM-2100. JEOL, Japan) was used to observe the morphology of the engineered exosomes. Purified exosomes were transferred onto a carbon-coated grid, kept at room temperature for 20 min and then visualized under TEM.
To detect exosome-specific markers, three different exosomes pellets were lysed separately with RIPA buffer containing protease inhibitors (CoWin Biotechnology, China), isolated protein were quantified using Micro BCA protein assay kit, equal amount of protein samples were separated in SDS-PAGE, transferred to a PVDF membrane and blocked with blocking buffer. To detect the exosome protein CD63, AcGFP fusion protein and the presence of Cas9, the membrane was incubated with primary anti-CD63 (Cat#sc-5275, Santa cruz, 1:1000), anti-AcGFP (Cat#TA180011, ORIGENE, 1:1000) and anti-Flag (Cat#SAB4200071, Sigma-Aldrich, 1:1000) respectively, followed by HRP-conjugated anti-mouse IgG antibody (Cat#ab6728,Abcam, 1:500) and detected using a Tanon-4200 Chemiluminescent Imaging System.

The IgG and IgA antibody levels in mouse serum and BALF were measured by ELISA, as previously described^{55 (link)}. Briefly, ELISA plates were coated overnight at 4 °C with RBD protein of WT or BA.5 strain or HA protein of pH1N1 (100 ng/well, Sino Biological, Beijing, China). For lgG detection, serum samples were threefold serially diluted with 1:400 starting dilutions, and BALF samples were twofold serially diluted with 1:5 starting dilutions. For IgA detection, BALF samples were twofold serially diluted using an undiluted stock solution as the starting dilutions. Following 2-h incubation at 37 °C, secondary antibodies were added: HRP-conjugated anti-mouse IgG antibody (1:100000 dilution, ab6789; Abcam, Cambridge, UK); HRP-conjugated anti-mouse IgA (1:5000 dilution, 1040-05; Southern Biotech, Birmingham, AL, USA). Plates were again incubated for 1 h at 37 °C, followed by the addition of TMB substrate (NCM Biotech, Suzhou, China). Sulfuric acid (2 M H₂SO₄) solution was used to stop the reaction. The optical density (OD)_450–630 was recorded using a microplate reader (Tecan, Männedorf, Switzerland). The binding antibody endpoint titer was determined as the reciprocal of the highest serum dilution that yielded an absorbance greater or equal to 0.1 OD unit above the absorbance of the pre-immune samples.

Mouse kidney and spleen tissues were fixed in formalin and embedded in paraffin. H&E staining was used to assess the histological features of the kidney. The renal pathology was scored according to the criteria of previous studies (64 (link)). To assess the immune complex deposition in the kidney, we stained paraffin-embedded renal sections with rabbit anti-C3 antibody (Abcam) and HRP-conjugated anti-rabbit antibody (Abcam) for mouse C3 and HRP-conjugated anti-mouse IgG antibody (Abcam) for mouse IgG. PNA (GC zone), CD3 (T cell zone), and B220 (B cell zone) were used to determine the GC area. For PNA staining, spleen tissues were stained with biotinylated anti-peanut agglutinin (Vector Laboratories ), and then incubated with biotinylated anti-peanut agglutinin antibody (Vector Laboratories). For CD3 staining, rabbit anti-CD3 antibody (Abcam) and HRP-conjugated anti-rabbit antibody (Abcam) were used. For B220 (CD45R) staining, rat anti-mouse CD45R antibody (Abcam) and HRP-conjugated goat anti-rat IgG(H+L) antibody (Proteintech) were used. After incubation of primary antibody and HRP-conjugated antibody, the opal 7-Color Manual IHC Kit (Perkin Elmer) was used for fluorescence labeling. Images were captured by Perkin Elmer, and the images were analyzed by the Mantra system. Information on antibodies is provided in Supplemental Table 2.

The proteins in the swab extract or the proteins in the secreted mucus were denatured in reducing condition, separated by SDS-PAGE, and electroblotted to PVDF membranes (Millipore). Membranes were stained with AB solution pH 2.5 (Wako, Japan). After differentiation with 3% acetic acid followed by 100% methanol, the membrane was further stained using PAS staining kit (MUTO Pure Chemicals, Japan) according to the manufacturer’s instructions.
For the sialyl-Tn detection by Western blotting, the membranes were blocked with Carbo-Free blocking solution (SP-5040, Vector Laboratories), and incubated with the anti-Sialyl-Tn (clone MLS132, Cat.# 010-25881, FujiFilm, 1:1100 dilution) or anti-Muc5ac (clone 45M1, Cat.# ab3649, Abcam, 1:500 dilution), followed by the HRP-conjugated anti-mouse IgG antibody (Cat.# 7076, Cell Signaling Technology, 1:3000 dilution). After stripping, the same membrane was incubated with WGA-CF488A (Cat.# 29022-1, Biotium, 1:200 diliution). The ECL luminescence and the fluorescence were visualized with LAS-4000 (FujiFilm).

For the focus-forming assay, Vero cells were seeded in 24-well plates. Virus samples were added to the cells for 2 h at 37 °C and then incubated with 0.8% carboxymethylcellulose (CMC). After a 4-day incubation, cell monolayers were fixed with 3.7% formaldehyde (FA) in PBS, followed by permeabilization with 0.15% Triton X-100 in PBS for 5 min. Cells were incubated with a mouse anti-GFP (Abcam, Cambridge, UK) at dilution 1:1000 followed by incubation with HRP-conjugated anti-mouse IgG antibody (Abcam, Cambridge, UK) at dilution 1:2000. Virus plaques were revealed with Vector NovaRED^TM peroxidase substrate (Cliniscences Nanterre, France) and counterstained with 0.5% crystal violet in 20% ethanol. Plates were scanned with an Epson scanner (Levallois-Perret, France).