A21436

Early and mid-gestation hfBECs (N = 3 subjects in duplicates/group) were cultured to approximately 75% confluence specifically for immunofluorescence analysis as described previously [38 (link)]. In brief, hfBECs were rinsed with cold PBS, fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 15 min and then permeabilized with 0.05% Tween 20 in PBS (5 min, room temperature). Autofluorescence was reduced using 0.1% Sudan Black in 70% ethanol (1 min) and non‐specific binding was blocked using 2% BSA for 1 h. Slides with hfBECs were incubated with primary antibodies, TLR-3 (ab62566, 1:500, Abcam, Toronto, ON, Canada), TLR-4 (ab22048, 1;200, Abcam), TLR-8 (ab180610, 1:200, Abcam), von Willebrand Factor (vWF) (ab11713,1:500, Abcam), anti-rabbit IgG (ab171870, 1:500, Abcam) and anti-mouse IgG2b (X0944D,1:500, Dako, Burloak, ON, Canada). overnight at 4 °C. Slides were then incubated with fluorescent secondary antibodies; the anti-mouse Alexa 488 (A21202, 1:1000), the anti-rabbit Alexa 488 (A21206, 1:1000), the anti-rabbit Alexa 568 (A10042, 1:1000), or the anti-sheep Alexa 555 (A21436, 1:000) secondary antibodies (Thermo Fisher Scientific) and counterstained with DAPI (1 μg/mL, 1 h). Fluorescent microscopy was performed using a spinning disc confocal microscope at various magnification (Leica DMI6000 B, Concord, ON, Canada).

Popliteal arteries were fixed with 4% paraformaldehyde for 24 h at room temperature, followed by procession by a serial alcohol gradient and embedment in paraffin wax blocks. The slides were incubated with primary antibodies that include GAB1 Rabbit mAb (#3232; Cell Signaling), LC3B (E5Q2K) Mouse mAb (#83506; Cell Signaling), and Human CD31/PECAM-1 Antibody Sheep mAb (#AF806-SP; R&D) overnight at 4°C, followed by the incubation of secondary antibodies that include donkey anti-rabbit IgG Alexa 488 (#A32790, Invitrogen, green), donkey anti-mouse IgG Alexa plus 647 (#A32787, Invitrogen, pink), and donkey anti-Sheep IgG Alexa 555 (#A-21436, Invitrogen, red). Nuclei were stained with DAPI (#D1306; Invitrogen, Paisley, United Kingdom). An epifluorescence microscope (#DM4000 M; Leica, Wetzlar, Germany) was used for image capture and analysis.