Alexa fluor 488 and 647

200 µg of antibody solutions (in PBS 1X) were mixed with N-Hydroxysuccinimide (NHS) ester functionalized dye (dye/protein ratio set to 3:1) with sodium bicarbonate solution 0.1 M (pH 8.3). We used Alexa Fluor 488 and 647 (Thermofisher Scientific) or Atto 565 (Sigma Aldrich). After incubation for 1 h at RT under constant agitation, uncoupled dyes were separated from labeled antibodies by size exclusion chromatography (on Sephadex G25 PD10 columns GE Healthcare) followed by dialysis against PBS 1 × overnight at 4 °C. At last, a dialysis against PBS 1 × was performed for 1 hr. The concentration and labeling efficiency was then assessed on a Nanodrop 100 (Thermo Scientific). The obtained dye/protein ratio was typically between 1 and 2.

Antibodies used were as follows: Notch-1 (4380T, Cell Signaling), DLL-1 (ab85346, Abcam), RBPjk (ab180588, Abcam), TAL-1 (PA5-30586, Invitrogen), GAPDH (GTX100118, GeneTex), DENV envelope monoclonal antibody D1-4G2-4-15 (HB-112, ATCC). HRP-conjugated secondary antibodies were purchased from Jackson Immunochemicals. Fluorescence-conjugated Alexa fluor 488 and 647, and Annexin V-alexa 488 were from Invitrogen. FACS antibodies CD61-APC and CD42a-Alexa 488 were from BD Biosciences and CD41-PE from Biolegend.

Platelets treated with anti-GPIbα or anti-GPIIbIIIa antibodies as described above were fixed in 4% paraformaldehyde at 22 °C for 20 min and spun down onto poly-L-lysine-coated coverslips (BD Biosciences; 500g, 5 min). Cells were blocked overnight in PBS with 1% BSA at 4 °C. Cells were incubated with primary antibodies rabbit anti-NEU1 IgG (4 μg ml⁻¹) (Santa Cruz) and our anti-CD61 mAb M1 (5 μg ml⁻¹) overnight at 4 °C. The platelets were washed in triplicate with PBS before addition of species appropriate secondary antibodies Alexa Fluor 488 and 647 (1:10,000) (Invitrogen). Images were taken with a Zeiss LSM 700 Confocal laser scanning microscope with a × 63 objective. Images were analysed with ZEN 2010 software (Zeiss) and Adobe Photoshop 7.

Cells were grown in µ-slide eight wells (Ibidi). Cells were fixed with 4% PFA and permeabilized with 0.5% Triton X-100 in PBS. Immunostaining was performed with primary antibodies for 1 h at room temperature for HA (1:1,000; BioLegend) or rabbit anti-IFITM3 (Cat. no. AP1153a, 1:500; Abcepta) and secondary antibodies conjugated to Alexa Fluor 488 and 647 (1:1,000; Invitrogen) for 1 h at room temperature. Nuclear DNA was stained with 2.5 µg/ml DAPI (Invitrogen) for 5 min at room temperature. Microscopic analysis was performed using a Nikon Ti-E microscope equipped with a Yokogawa CSU-X1 spinning-disc and an Andor DU-888 camera. ImageJ was used to prepare microscopy images and for quantification of signal intensity of the immunostaining.

Sections were washed with PBS for 5 min, then permeabilized and blocked in a PBS solution containing 1% BSA, 0.3% Triton X-100, 2% (v/v) donkey serum, and 0.02% sodium azide for 30 min at room temperature. Sections were incubated with primary antibodies in blocking solution overnight at 4°C. Primary antibodies include anti-MCHR1 (rabbit pAB; 1:250 dilution; catalog #711649, Thermo Fisher Scientific), anti-adenylate cyclase 3 [ACIII; 1:1000 dilution; chicken polyclonal antibody (pAb); CPCA-ACIII, Encor], anti-mCherry (chicken pAb; 1:1000 dilution; catalog NBP2-25158, Novus), anti-MCH (1:200 dilution; rabbit mAb; catalog #274415, Abcam). Sections were then washed with PBS before incubating with secondary antibodies for 1 h at room temperature. Secondary antibodies include donkey conjugated Alexa Fluor 647 and 488 (1:1000; Thermo Fisher Scientific) against appropriate species according to the corresponding primary. All primary and secondary solutions were made in the blocking solution described above. Slides were then washed in PBS and stained with Hoechst nuclear stain (catalog #H3570, Thermo Fisher Scientific) for 5 min at room temperature. Coverslips were mounted using SlowFade Diamond Antifade Mountant (catalog #S36972, Thermo Fisher Scientific).