Stable diaminobenzidine dab

Peripheral blood mononuclear cells (PBMCs) were collected from study persons prior to (days 0 and 28) and 7 days after each vaccine dose (days 7 and 35) for measurement of ASCs. Cryopreserved PBMCS were used for determining the number of ASCs by enzyme-linked immunosorbent spot (ELISPOT) assays as described previously (18 (link)). Briefly, 96-well polyvinylidene difluoride (PVDF) membrane plates (Millipore) were coated with 1 μg per well of GI.1 or GII.4 (consensus) VLPs or phosphate-buffered saline (PBS) overnight at 4°C. Cryopreserved PBMCs were thawed, and doubling dilutions were prepared in AIM-V medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), sodium pyruvate (Invitrogen), minimum nonessential amino acids (Invitrogen), and β-mercaptoethanol (Invitrogen), starting with 5 × 10⁵ PBMCs per well in the first row. Following incubation in a humidified incubator with 5% CO₂ at 37°C for about 18 h, IgA and IgG ASCs were detected using goat anti-human IgA and IgG antibodies conjugated to horseradish peroxidase (HRP), respectively (Southern Biotech), and stable diaminobenzidine (DAB) as a substrate (Invitrogen). The membrane was allowed to dry overnight, and spots were imaged using an ImmunoSpot reader (Cellular Technology Limited).

Tissue distribution of the C3 protein was detected by IHC staining using light microscopy, as previously described^{51 (link)}. Briefly, the mid colon tissue samples were fixed in 10% formalin for 12 h, embedded in paraffin, and sliced into 4 µm thick sections. These sections were subsequently deparaffinized with xylene, rehydrated, and pretreated for 30 min at room temperature with 1× PBS blocking buffer containing 10% goat serum (Vector Laboratories, Burlingame, CA, USA). The sections were then incubated with primary anti-C3 antibody (Abcam Com., Cambridge, UK), diluted 1:300 in 1× PBS blocking buffer. Also, a group treated with only HRP-conjugated secondary antibody without primary antibody was analyzed as control to rule out false signals during the immunohistochemical analysis. The antigen–antibody complexes were visualized with biotinylated secondary antibody (goat anti rabbit)-conjugated horseradish peroxidase (HRP) streptavidin (Histostain-Plus Kit, Zymed, South San Francisco, CA, USA), at a dilution of 1:300 in PBS blocking buffer. Finally, C3 proteins were detected using stable diaminobenzidine (DAB) (Invitrogen Co., Carlsbad, CA, USA) and the Leica Application Suite (Leica Microsystems, Wetzlar, Germany).

The tissue distribution of nuclear factor erythroid 2–related factor 2 (Nrf2), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) proteins were detected by IHC as described in a previous study [32 (link)]. After deparaffinization of tissue sections, anti-Nrf2 (Abcam, Cambridge, UK), anti-COX-2 (Cell Signaling Technology Inc., Danvers, MA, USA), anti-iNOS (Thermo Fisher Scientific Inc., Waltham, MA, USA) and goat alkaline phosphatase (AP) conjugated anti-rabbit IgG (1:200, Thermo Fisher Scientific Inc., Waltham, MA, USA), antibodies were sequentially treated onto these sections. Finally, the distribution of each protein in the retina was detected using stable diaminobenzidine (DAB) (Invitrogen Co., Waltham, MA, USA) and evaluated using the Leica Application Suite (Leica Microsystems).