Rat anti mouse sca1

Using cytospin, the bone marrow mononuclear cells were prepared on slides and fixed in 4% paraformaldehyde. The smear was permeabilized in 0.2% Triton and incubated with the following monoclonal antibodies: 1:100 rabbit anti-mouse α7nAChR and 1:100 rat anti-mouse Sca1 (BD Pharmingen). Then, anti-rabbit or rat Fluor 594 or FITC-labeled secondary antibody (Molecular Probes) was added to the smear and incubated for 1 h. After washing with hypertonic PBS (2.7% NaCl), the slides were mounted with ProLong Gold antifade reagent (Molecular Probes). For the bone marrow immunofluorescence, we carefully harvested the bone marrow, embedded it in OCT (Sakura FineTek, Torrance, CA, USA), and froze it in liquid nitrogen. Frozen sections (5 μm) were prepared in a cryostat (MICROM HM 505E). The sections were incubated with anti-α7nAChR, VE-cadherin, and p-Akt1 fluorescent antibodies and corresponding secondary fluorescent antibodies. All staining procedures were performed with appropriate isotype controls.

The cell surface markers of MSCs were analyzed using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). Briefly, cells that reached 90% confluence were harvested using 0.25% EDTA and washed twice in Dulbecco’s phosphate buffered saline supplemented with 10% FBS. The cells for detecting CD11b, CD34, CD45, Sca-1, CD44 and CD73 were labeled directly with BB515 or PE-conjugated CD markers (rat anti-mouse CD11b [1: 100, BD Pharmingen; BD Biosciences, Franklin Lake, NJ, USA], rat anti-mouse CD34 [1: 100, BD Pharmingen], rat anti-mouse CD45 [1: 100, BD Pharmingen], rat anti-mouse Sca-1 [1: 100, BD Pharmingen], rat anti-mouse CD44 [1: 100, BD Pharmingen], rat anti-mouse CD73 [1: 100, BD Pharmingen]).