Dapi containing medium

Immunofluorescent detection of NFATc1 was performed according to the method described by Hasega et al. 25 with minor modifications. Briefly, cells were plated at less than 50% confluence (a density of 5 × 10⁴ cells/chamber) in a four‐chamber slide (Nunc, Rochester, NY, USA), grown overnight, and treated with vehicle or drugs for 24 h in complete α‐MEM. After fixation with 4% paraformaldehyde for 10 min at room temperature, cells were permeabilized with 0.2% Triton X‐100 for 15 min and then blocked with 5% bovine serum albumin at room temperature for 1 h. After washing and blocking, anti‐NFATc1 antibody was added at a dilution of 1 : 100 and the fixed cells were incubated at 4 °C overnight. NFATc1 signals were detected with Alexa Fluor 488‐conjugated secondary antibody (Invitrogen) at a dilution of 1 : 500 for 1 h, after which the fixed cells were mounted in DAPI‐containing medium (Sigma).

Mice were sacrificed by cervical dislocation and femurs dissected for fixation in PFA. Fixed femurs were then sliced into 7μm sections on Kawamoto adhesive film and attached to superfrost glass slides as described previously [13 (link)]. For immunofluorescence staining sections were thawed and rehydrated in PBS. After blocking with 5% FCS/0.1% Tween-20 sections were probed with primary antibodies against CD105 (eBioscience, San Diego, CA, USA), Endomucin (Santa Cruz, Dallas, TX, USA), laminin α5 (clone 504) [14 (link)] collagen type I (ab21286) and β₁-tubulin (Italiano laboratory, Boston, MA, USA). Corresponding secondary antibodies conjugated with Alexa Fluor 546 (A-110819) or Alexa Fluor 647 (A-21244, A-21247) (all Invitrogen, Carlsbad, CA, USA) were used to detect IgG of rat, rabbit, or mouse origin. Megakaryocytes were stained with an Alexa Fluor 488-coupled antibody raised against GPIX (clone 56F8, Nieswandt laboratory, Würzburg, Germany). For detection of F-actin Alexa Fluor 647-conjugated phalloidin (Invitrogen) was used. All slides were mounted with DAPI-containing medium (Sigma-Aldrich, St. Louis, MO, USA) to visualize nuclei.