American Society of Hematology

Figure 5

$Figure 5. PAR1 expression by stromal niche cells. (A) A representative immunohistological staining of frozen femur from C57BL/6 mouse is shown. Sample was stained for Osteopontin (red) and PAR1 (green). Arrowheads point to some of the positive PAR1 staining. PAR1-positive cells were detected within the bone trabeculae, along the endosteum and as scattered cells areas inside the hematopoietic compartment (magnification, ×10). (B) Two larger magnification (×40) samples of staining for extracellular OPN together with membrane bound PAR1 staining are shown. Isotype control staining is depicted at the inset, using an irrelevant isotype-matched primary Ab for both PAR1 and OPN, followed by the secondary Ab used for the specific staining. OPN, osteopontin. (C) PAR1 specific expression on osteoblasts population is shown. Osteocalcin (green) is used as osteoblast specific intracellular marker. PAR1 is represented in red. Merge (yellow) demonstrates PAR1 expression on osteocalcin positive osteoblasts. Nuclei are denoted by Hoechst staining (blue). (D) FACS analysis of PAR1 expression on stromal cells. PAR1-positive signal was detected on CD45−CD31−Ter119− + Sca1−PDGFRα+ subpopulation in C57BL/6 mice (red line). Similar cells of PAR1 KO mice were used as negative control (green line). (E) PAR1 expression was determined in GFP-CXCL12 and GFP-Nestin mice. Flushed fraction of BM was stained for CD45, CD31, and Ter119 and the negative fraction was examined for GFP signal. As can be seen, PAR1 expression was determined in both GFP CXCL12 (green) and GFP-Nestin (blue) mice. Isotype antibody staining was used as a negative control.$

PAR1 expression by stromal niche cells. (A) A representative immunohistological staining of frozen femur from C57BL/6 mouse is shown. Sample was stained for Osteopontin (red) and PAR1 (green). Arrowheads point to some of the positive PAR1 staining. PAR1-positive cells were detected within the bone trabeculae, along the endosteum and as scattered cells areas inside the hematopoietic compartment (magnification, ×10). (B) Two larger magnification (×40) samples of staining for extracellular OPN together with membrane bound PAR1 staining are shown. Isotype control staining is depicted at the inset, using an irrelevant isotype-matched primary Ab for both PAR1 and OPN, followed by the secondary Ab used for the specific staining. OPN, osteopontin. (C) PAR1 specific expression on osteoblasts population is shown. Osteocalcin (green) is used as osteoblast specific intracellular marker. PAR1 is represented in red. Merge (yellow) demonstrates PAR1 expression on osteocalcin positive osteoblasts. Nuclei are denoted by Hoechst staining (blue). (D) FACS analysis of PAR1 expression on stromal cells. PAR1-positive signal was detected on CD45⁻CD31⁻Ter119⁻ + Sca1⁻PDGFRα⁺ subpopulation in C57BL/6 mice (red line). Similar cells of PAR1 KO mice were used as negative control (green line). (E) PAR1 expression was determined in GFP-CXCL12 and GFP-Nestin mice. Flushed fraction of BM was stained for CD45, CD31, and Ter119 and the negative fraction was examined for GFP signal. As can be seen, PAR1 expression was determined in both GFP CXCL12 (green) and GFP-Nestin (blue) mice. Isotype antibody staining was used as a negative control.

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