Hpa004334

Anti-human protein antibodies used for platelet and imMKCL cell IB and IF staining were as follows: rabbit polyclonals to ARPC1A (Sigma-Aldrich, HPA004334; dilutions 1/250 or 1/500 for IB, 1/25 for IF), ARPC1B (Sigma-Aldrich, HPA004832; dilutions 1/100 or 1/500 for IB, 1/100 for IF), ARPC2/p34ARC (Millipore, 07-227; dilution 1/1,000 for IB), ARPC3 (Sigma-Aldrich, HPA006550; dilution 1/1,000 for IB), ARPC5/p16ARC (Abcam, ab118459; dilutions 1/1,000 for IB, 1/100 for IF) and α-tubulin (Cell Signaling, 11H10; dilutions 1/1,000 for IB, 1/100 for IF); rabbit monoclonals to ARP2 (Abcam, ab128934; dilution 1/1,000 for IB), ARP3 (Abcam, ab151729; dilution 1/1,000 for IB) and WASP (Cell Signaling, D9C8; dilution 1/1,000 for IB); mouse monoclonals to γ-tubulin (Thermo Scientific, 4D11), α-tubulin (Sigma-Aldrich, Clone B-5-1-2; dilutions 1/1,000 for IB, 1/100 for IF), CD61 (Dako, Clone Y2/51; dilution 1/200 for IF), TSP1 (R&D Systems, clone 301221; dilution 1/100 for IF) and GAPDH (Millipore, MAB374; dilution 1/5,000 for IB), and a goat polyclonal to P-selectin (Santa Cruz, sc-6941; dilution 1/100 for IF).

Paraffin sections of colon and skin were deparaffinized for 5 min each with: xylene twice, 100% ethanol twice, 95% ethanol twice, 75% ethanol, ddH2O. Antigen retrieval was performed with a Decloaking Chamber (NxGen) using a buffer containing 1.27 mM EDTA, 3.75 mM boric acid, 0.61 mM sodium borate and 0.003% ProClin. The slides were then blocked for 1 h (23 °C) with 5% BSA and 15% donkey serum in PBS. ARPC1B antibody (Sigma-Aldrich, HPA004832; dilution 1/100 in blocking buffer) kept overnight at 4 °C in a humidified staining chamber. After washing the slides three times with PBS containing 0.05% Tween 20, they were stained with Alexa Fluor 594 donkey anti-rabbit Fab fragment (Jackson ImmunoResearch; dilution 1/250) for 1 h at (23 °C). Slides were blocked with donkey anti-rabbit Fab fragment (Jackson ImmunoResearch; dilution 1/100) for 1 h (23 °C), then incubated with polyclonal rabbit anti-ARPC1A (Sigma-Aldrich, HPA004334; dilution 1/25) overnight at 4 °C. Secondary Alexa Fluor 488 donkey anti-rabbit (Jackson ImmunoResearch; dilution 1/250) was added for 1 h at RT. To minimize autofluorescence the slides were treated with 3.3 mM Sudan Black in 70% ethanol for 10 min (23 °C). Slides were then stained with DAPI (dilution 1/5,000) and mounted with Dako fluorescent mounting medium (S3023).