Collagen type 1

Fertilised hen’s eggs were incubated at 38 °C until HH stage 11 or stage 13 [24 (link)]. Embryos were dissected and extra-embryonic tissue removed. For the pharmacological manipulation of signalling pathways, embryos were cut anterior to the midbrain/diencephalon border and posterior of the otic vesicle. For the midbrain tissue explant cultures, the midbrain region was first dissected and the isthmus or roof plate regions removed using fine tungsten needles. Explant pieces were embedded in type I collagen (Roche), dorsal side up and cultured overnight, at 37 °C in F12 medium (Sigma) supplemented with 10% fetal calf serum (Life Technologies) and penicillin/streptomycin (Sigma). All drugs were dissolved in DMSO to the following stock concentrations: dorsomorphin, 5 mM; SU5402, 34 mM; IWP-2; 1 mM. Stock solutions were stored at −20 °C. Drugs, or equal volume of DMSO (1-9ul /ml depending on the corresponding concertation of the drug) for the controls, were added to the culture medium. Explants were fixed in formaldehyde for subsequent immunostaining.

The 96-well microtiter plates were coated (overnight at 4 °C) by adding 100 μL per well either with collagen type I (100 μg/mL; Roche Diagnostic S.p.a. Monza, Monza Brianza, Italy) or human fibrinogen (100 μg/mL; Merck Life Science S.r.l.). The non-specific platelet adhesion was prevented by the pre-treatment of the wells with 200 μL 1% bovine serum albumin for 1 h at 37° C. Following three washing steps, 50 μL PRP (treated with 100 μg/mL NAC or vehicle for 30 min at 37 °C) were added to each well and incubated at 37 °C without stirring to allow platelet adhesion. After 1 h, non-adherent platelets were removed, the plate was washed, and adherent cells were quantified through the measurement of the acid phosphatase activity. Briefly, 150 μL of acid phosphatase substrate (100 mmol/L citrate buffer pH 5.4, containing 5 mmol/L p-nitrophenyl phosphate and 0.1% Triton X-100) was added to each well. After 1 h incubation at 37 °C, the reaction was stopped by the addition of 100 μL NaOH 2 N. The p-nitrophenol produced was then measured by reading the absorbance at 405 nm (Infinite M Plex, Tecan, Tecan S.r.l., Cernusco sul Naviglio, Milan, Italy). A set of experiments was performed using tirofiban (Sigma-Aldrich S.r.l.), an αIIbβ3 antagonist. PRP was treated with tirofiban 5 μg/mL, for 30 min at 37 °C before the adhesion assay.