TLT-1 appears more rapidly and is detected throughout laser-induced thrombi in vivo. Mice were injected with anti-GPIbβ, anti-TLT-1, or anti-CD62P antibody. Arterioles of cremaster muscles were subsequently injured by laser. (A) Representative composite bright-field and fluorescence images of platelets (GPIbβ), TLT-1, and P-selectin in thrombi. Scale bars, 10 µm. (B) Quantification of fluorescence area in pixels (data represent mean ± standard error of the mean; n = 25-32 injuries from 4 to 6 mice). (C) Distinct and overlapping staining of TLT-1 and P-selectin in platelets and megakaryocytes. Resting human (top row) and mouse (middle row) platelets seeded on poly-l-lysine and primary mouse BM-derived megakaryocytes (MK) (bottom row) spread on fibrinogen matrix for 15 minutes at 37°C were fixed, permeabilized, and stained with Alexa Fluor 488 anti-TLT-1 and Alexa Fluor 647 anti-P-selectin antibodies. The right column represented an overlay of both images. Scale bars, 5 µm. Images are representative of 3 to 4 independent experiments. (D) Degree of TLT-1 and P-selectin colocalization as determined by the Manders overlap coefficients (M1: TLT-1:P-selectin and M2: P-selectin:TLT-1) in resting human (i) and mouse platelets (ii), and object-based colocalization in mouse BM-derived megakaryocytes (iii) (n = 3-4 independent experiments per condition, 400-600 platelets, and 25-35 megakaryocytes per experiment).