Vector vip peroxidase substrate kit

DENV-1 KDH0026A strain was kindly provided by Dr. Lambrechts (Institut Pasteur, France). Mouse-adapted DENV-2 S221 strain was kindly provided by Dr. Shresta (La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA). DENV-3 PaH881/88 and DENV-4 ThD4_0087_77 strains were both isolated in Thailand in 1988 and 1977, respectively. Virus stocks were produced in Vero E6 cells grown in T-175 tissue flasks with filter cups. Titrations were performed on Vero E6 cells grown on 24 well plates. To do so, cells were inoculated with 300µl of serial stock dilutions for 1 h with periodic shaking. After removal of the inoculation medium, cells were overlayed with DMEM containing 1.6% carboxymethyl cellulose, 2% of heat-inactivated fetal calf serum (FBS), and 1% penicillin/streptomycin. At day 5 post-inoculation, the overlaying medium was removed, cells were fixed with 4% paraformaldehyde for 30 min, and viral foci were revealed by staining with 0.5µg/mL of 4G2 mouse-anti-DENV envelope protein antibody, produced by the recombinant protein production facility of the Institut Pasteur, followed by the second staining with goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) (BioRad, France). The HPR signal was revealed with the Vector VIP peroxidase substrate kit (Vector Laboratories, USA) following the manufacturer’s recommendations.

Viral focus-forming assays were performed on VERO E6 cells. Briefly, Vero E6 cell monolayers (1 × 10⁵ cells/well) in 24-well tissue-culture trays were inoculated in duplicate with 200μL 10-fold serial dilutions of each sample and incubated for 2 hours at 37 °C. Unadsorbed virus was removed, after which 1 ml of DMEM supplemented with 1.6% carboxymethyl cellulose (CMC) 2% FCS was added to each well and incubated for 3 days. The CMC overlay was aspirated, and the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.1% Triton X-100 for 3 min. After fixation, the cells were washed with PBS and incubated for 1 h at room temperature with anti-E antibody (4G2), followed by incubation with HRP-conjugated anti-mouse IgG antibody. The assays were developed with the Vector VIP peroxidase substrate kit (Vector Laboratories) according to the manufacturer's instructions. The viral titers were expressed in focus-forming units (ffu) per milliliter.

Vero-E6 cells were seeded at 8 × 10⁴ cells per well in 24-well plates and incubated at 37°C for 24 h. Tenfold dilutions of virus in DMEM were added to the cells and incubated for 1 h at 37°C. Unadsorbed virus was removed, and then 1 ml of DMEM supplemented with 1.6% carboxymethyl cellulose (CMC), 10 mM HEPES buffer, 72 mM sodium bicarbonate, and 2% FBS was added to each well, followed by incubation at 37°C for 2 days. The CMC overlay was removed, and the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.2% Triton X-100 for 5 min. Cells were then washed with PBS and incubated for 1 h at room temperature with anti-E antibody (4G2), followed by incubation with HRP-conjugated anti-mouse IgG antibody. The foci were revealed using the Vector VIP peroxidase substrate kit (Vector Laboratories) according to the manufacturer’s instructions.

To localize pNrf2 in the kidney, double staining was performed to identify pNrf2 positives in different types of kidney cells. Staining of the first antigen (pNrf2) was performed as described above using diaminobenzidine (DAB) as a chromogen. After visualization of antibody binding, the protocol was repeated with an antibody against calbindin (1:200, #2173, Cell Signaling, Herts, UK) to identify distal tubular cells and cells of the early collecting duct. Proximal tubular cells were identified by the presence of the brush border, whereas glomeruli were identified by their capillary tuft (blue circles). Fifty glomeruli were analyzed per animal. The late collecting duct was identified by the absence of positive calbindin staining and brush border. The VECTOR^® VIP Peroxidase Substrate Kit was used to visualize the second antigen (SK-4600, Vector Lab, Burlingame, CA, USA), which produced a purple stain. Sections were counterstained with hematoxylin and dehydrated in descending alcohol concentrations. Pictures were taken at 400-fold magnification. The ratio of positive to negative nuclei for pNrf2 on 10 visual fields was assessed via ImageJ [30 (link)] by counting only nuclei positive for the specific kidney cell identifier.

Immunohistochemistry (IHC) was performed using Impress Peroxidase/Alkaline Detection Reagents (Vector Laboratories) and antibodies specific for GLUT1 (Abcam, ab115730), LC3B (Proteintech, 18725-1-AP), /PLIN2 (Proteintech, 15294-1-AP), and LAL (Novus, NBPI-54155SS). Double immunostaining was performed as described previously [28 (link)]. Color reaction was developed with the VECTOR VIP Peroxidase Substrate Kit, (Vector Laboratories) or HighDef^® red IHC AP chromogen Enzo. Image quantification was performed using NIH ImageJ1.x software.

BHK-21 cells were seeded in 24-well plates. Tenfold dilutions of virus samples were prepared in duplicate in DMEM and 200 μl of each dilution was added to the cells. The plates were incubated for 1 h at 37°C. Unadsorbed virus was removed, after which 1 ml of DMEM supplemented with antibiotics and antifungals, 1.6% carboxymethyl cellulose (CMC), 10 mM HEPES buffer, 72 mM sodium bicarbonate, and 2% FBS was added to each well, followed by incubation at 37°C for 32 h. The CMC overlay was aspirated, and the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.1% Triton-X100 for 5 min. After fixation, the cells were washed with PBS and incubated for 1 h at room temperature with anti-E antibody (4G2), followed by incubation with HRP-conjugated anti-mouse IgG antibody. The assays were developed with the Vector VIP peroxidase substrate kit (Vector Laboratories) according to the manufacturer’s instructions. The viral titers were expressed as focus forming units (FFU)/ml.