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. 2020 Mar 18;6(12):eaay6314. doi: 10.1126/sciadv.aay6314

Fig. 1. Platelet microvascular deposition and MC activation.

Fig. 1

(A) Hematoxylin and eosin staining of rat colons 2 hours after completion of the DHCA model. Arrow indicates platelet-rich thrombus in a small submucosal vessel. Immunofluorescence staining for CD41 (green) in sham (B) or experimental (C and D) animals 2 hours after completion of DHCA model (4′,6 diamidino-2-phenylindole nuclear counterstain). Representative images of n = 4 per condition; magnification, ×200. (D) Insert in (C) in 600×. (E) Z-stack confocal laser scanning micrographs of whole-mount sections (ear) taken from Mcpt5-Cre tdTomatofl/fl mice 60 min after intravenous injection of a collagen and epinephrine mixture. Endogenously expressed tdTomato (red) outlines MCs with additional staining performed against CD41 (green) and CD31 (blue). An animated three-dimensional reconstruction can be viewed in the Supplementary Materials. (F) MC granule staining [tetramethyl rhodamine isothiocyanate (TRITC)–avidin, red; arrows indicate released MC granules] reveals activated MCs in close vicinity to intravascular (anti-CD31, blue) platelet aggregates (anti-CD41, green). Representative images of n = 4 per condition. (G) Rectal temperature following collagen and epinephrine injection (CollE) in comparison to anaphylaxis after sensitization with trinitrophenol (TNP)–specific IgE and exposure to TNP-conjugated ovalbumin (TNP OVA) or vehicle control treatment. In addition, platelets were activated with a monoclonal antibody against mouse integrin αIIb, clone MWReg30 (MWReg). A subset of animals was platelet-depleted before receiving collagen and epinephrine (CollE Plt dplt) or MWReg (MWReg Plt dplt). n = 6 per condition. *P < 0.05 versus vehicle control, two-way ANOVA. (H) Plasma chymase levels following collagen and epinephrine, or MWReg injection or TNP OVA anaphylaxis. Data are represented as the means ± SD. n = 4 to 6 per condition. *P < 0.05 versus vehicle control and #P < 0.05 versus respective treatment group, one-way ANOVA and Tukey’s multiple comparisons test.