Vector vip

4 µm sections of paraffin-embedded tumors and TMA were dried at 56°C and then treated twice with xylene for 10 min at room temperature. Subsequently, sections were washed twice with absolute ethanol and twice with 70% ethanol followed by one rinse with bi-distilled water. For antigen retrieval, sections were incubated with citrate buffer pH 9.0 (DAKO, Hamburg, Germany) for 10 min at 90°C and rinsed with bi-distilled water. Next, slides were rinsed twice with phosphate-buffered saline (PBS) and thereafter incubated with Blocking Solution (DAKO, S2023) for 10 min at room temperature. After two additional washing steps with PBS for 10 min at room temperature, the monoclonal α-p53 antibody (DO-7, DAKO) or α-TRP2 antibody (D18, Santa Cruz) was added to the sections at a predetermined concentration in PBS, followed by an over night incubation at 4°C. After two 10 min washes in PBS, biotinylated multispecies-specific secondary antibody (DAKO, K5003) was added to the sections for 30 min at room temperature. Slides were then washed twice in PBS/bovine serum albumin, and bound antibodies were visualized using streptavidin-HRP (DAKO K5003) and Vector Vip (Vector Laboratories, Burlingame USA) as peroxidase substrate according to the manufacturer's guidelines. Finally, the nuclei were stained with hemalaun.

Samples were serial diluted in DMEM–2% FBS and applied onto 90% confluence Vero cell monolayers for 2 h at 37 °C, 5% CO2. After viral adsorption, inocula were removed and replaced by a mixed solution of DMEM–4% FBS and PBS–1% carboxymethyl cellulose in a 1:1 ratio for 3 days. After 3 days, cells were fixated in PBS–4% paraformaldehyde for 15 min at room temperature (RT), and permeabilized with PBS–0.1% Triton for 3 min at RT. Non-specific sites were blocked with PBS–1% bovine serum albumin–0.1% Tween 20 for 30 min at RT. ZIKV envelope protein (E) staining was performed using the mouse anti-E (clone 4G2) antibody as the primary antibody and the horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Biorad, Hercules, CA, USA) as the secondary antibody. Finally, freshly prepared peroxidase substrate (Vector Vip; Vector Laboratories) was added for 5–15 min and the foci of infection were manually counted.