Hrp dab chromogenic substrate kit

After the centrifugation of the disrupted CeNVD_pET-28a(+), the cleared supernatant and precipitant were loaded on SDS–PAGE gels, then all the protein molecules was transferred to a PVDF membrane and blocked in a PBST buffer (PBS pH 8.0, 0.02% Tween-20) containing a 1% bovine serum albumin (BSA) for 2 h, followed by incubation in an anti-His-tag mouse monoclonal antibody (Abcam, Cambridge, United Kingdom), which was diluted in a blocking buffer (PBST pH 8.0, 1% BSA) at the indicated concentrations of 1:5,000 for 12 h at 4°C. After washing with PBST for four times, the membrane was protected from light and incubated with the HRP-conjugated secondary antibody (HRP-conjugated goat anti-mouse IgG, Tiangen Biochemical Technology, Beijing, China) at a dilution of 1:1,000 and room temperature for 2 h. After washing, the target protein was trapped by the HRP-DAB chromogenic substrate kit (Tiangen Biochemical Technology, Beijing, China), and the immunoreactive band was digitally scanned using an Odyssey Infrared Imager (LI-COR Bio-science, Lincoln, NE, United States) (Wan et al., 2016 (link)).

Grouper spleen cells were collected and lysed in Pierce IP Lysis Buffer (Thermo Scientific, United States). Proteins were separated by 12% SDS-PAGE and transferred onto Immobilon-polyvinylidene difluoride membranes (Millipore, Temecula, CA, United States). Blots were incubated in 5% skim milk for 2 h, and were incubated with indicated primary antibodies: anti-Rab5c (1:500 dilution), anti-β-tubulin (1:2,000 dilution), anti-SGIV major capsid protein (MCP; 1:1,000 dilution), and anti-LC3 (1:1,000 dilution). After washing three times in PBS Tween membranes were incubated with peroxidase-conjugated affinipure goat anti-rabbit IgG (1:5,000 dilution). Immuno-reactive proteins were visualized using an enhanced HRP-DAB Chromogenic Substrate Kit (Tiangen, China) and SuperSignal (R) West Femto Trial Kit (Thermo Scientific, United States).

At 20 h.p.i., the infected mice were euthanized and the brains was collected and placed into 10% neutral buffered formalin. After fixation the organs were embedded in paraffin, 4 mm sections were taken, and these were stained with hematoxylin and eosin for histological evaluation. For immunohistochemistry, sequential slides were stained using an immunoperoxidase method as follows. First, sections were blocked with 3% H₂O₂ for 30 min, and then non-specific background staining was blocked by incubating the sections for 30 min with normal rabbit serum. The sections were then incubated for 1 h with rabbit anti-GBS polyclonal serum (1∶1000, prepared in our laboratory using the GBS strain GD201008-001 from this study) or unimmunized rabbit serum (1∶1000) as a control, followed by biotinylated goat anti-rabbit immunoglobulin (Ding-Guo, China) diluted 1/100 at 37°C for 30 min and subsequent incubation with HRP conjugated streptavidin (DingGuo, China) at 37°C for 30 min. Finally, the sections were developed with the HRP-DAB chromogenic substrate kit (Tiangen, China) for 10 min, and then the slides were counterstained with hematoxylin.