Super block

Sera was collected from mice 14 days post-primary and 14 days post-secondary injection and diluted according to the expected antibody response (between 1:10 and 1:5000). Plates were coated with 100 μl of the full-length M72 protein at 1 μg/mL. Following washing (PBS plus tween 20) and blocking (SuperBlock, Scytek Laboratories), plates were incubated with diluted serum for 1 h followed by anti-mouse IgG, IgG1, or IgG2a-HRP secondary antibody (Bethyl Laboratories) and TMB substrate (BD). Plates were read at 450 nm. Antibody titers were determined by calculating titer of each sample at OD 0.3.

Kidney sections were immunolabeled with primary abs against macrophages, using the anti-Iba1 (cat#NCNP24, Fujifilm Wako, Pure Chemical Corporation, Tokyo, Japan) and anti-cleaved casp-3 (cat#9664, Cell Signaling Technology, Danvers, MA, USA) diluted 1:400 in Super Block (cat#AAA125, ScyTek Laboratories, Logan, UT, USA). The slides were incubated with fluorescent secondary Goat anti-rabbit IgG H&L (Alexa Fluor^® 488; cat#ab150077, Abcam, Cambridge, MA, USA) diluted 1:400 in blocking solution. The slides were washed and mounted with DAPI-containing mounting medium, covered with glass coverslips, and imaged on a Sight DS-5M-L1 digital camera (Nikon, Melville, NY, USA) connected to an Eclipse 50i light microscope (Nikon) at different magnifications. The images acquired were used for quantifications of Iba1⁺ cells and mean fluorescence intensity (MFI) for cleaved casp-3 in renal tubular cells, using Image J software (NIH).

Blood was collected from mice 14 days post-vaccination, and serum was isolated and diluted according to the expected antigen-specific antibody titers (between 1:20 and 1:500). Nunc MaxiSorp plates were coated with 100 µL of CoaB peptide (GVIDTSAVESAITDGQGDM) at 10 µg/mL overnight at room temperature. Plates were washed with 1X PBS plus 0.05% tween 20 and then blocked with 200 µL of SuperBlock (Scytek Laboratories, Logan, UT, USA) for 2 h at 37 °C. Next, serially diluted serum at a dilution factor of 1:3 was added and incubated at 37 °C for 2 h, then washed three times with 1X PBS plus 0.05% tween-20. Following the washing step, plates were incubated with anti-mouse IgG, IgG1, IgG2b, or IgG2c-HRP secondary antibody at a dilution of 1:1000 (Southern Biotech, Birmingham, AL, USA) for 1 h at 37 °C and then washed the same as above. Plates were then incubated with 100 µL of TMB substrate (BD Biosciences, Franklin Lakes, NJ, USA) at room temperature for an hour, followed by absorbance being read at 650 nm using the Spectra Max 190 (Molecular Devices, San Jose, CA, USA). Antibody titers were determined using the XL fit software program version 5.5.05 (IDBS, Boston, MA, USA) at an OD of 0.3. Negative values were assigned a point value of 0.5 in order to plot points and follow up with statistical analysis.

The small intestines were formalin-fixed and paraffin-embedded. Antigen retrieval was performed on deparaffinized 5 micron sections which were then incubated for 5 min with Super Block (ScyTek #AAA999), and 10 min in 3% H₂O₂ to block the endogenous peroxidase activity. Sections were then incubated at 4°C overnight with polyclonal goat anti-rotavirus antiserum (NCDV; Meridian, LS; 1:500) or monoclonal rabbit anti-phospho-STAT1 (Tyr701; 58D6) (Cell Signaling; 1:500). Slides were washed 2 times in PBST (0.05% Tween-20 in PBS), then incubated at room temperature for 30 min with UltraTek anti-Goat biotinylated antibody (Ready to Use) (ScyTek #AGL125) or UltraTek anti-rabbit biotinylated antibody (Ready to Use) (ScyTek #ABK125), followed by a 20 min room temperature incubation with UltraTek Streptavidin/HRP (Ready to Use). NovaRED substrate solution (Vector, SK-4000) was used as a substrate. After immunostaining, tissue sections were washed twice in water and counterstained with Mayer’s haematoxylin and Scott’s bluing buffer.