Z1 fluorescent microscope

Protein extraction and Western blot analysis were performed as described (Schock et al., 2008 (link)). A custom rabbit antibody against IRF2BP2 was described previously (Teng et al., 2011 (link)). Cryostat sections (20 μm) were subjected to cresyl violet staining and immunofluorescence. Immunofluorescence images were acquired on a Zeiss Z1 fluorescent microscope. Primary antibodies used and their dilutions are: MHCII (Biolegend #107602, anti-rat 500× dilution), CD68 (Santa Cruz, sc70761, anti-mouse, 500×), CD206 (Santa Cruz, sc34577, anti-goat, 500×), Iba1 (WAKO, #019-19741, anti-rat, 500×), NeuN (Millipore MAB377, anti-mouse, 500×), and GFAP (Santa Cruz, sc170, anti-goat, 500×). Cy2-, cy3-, cy5-conjugated secondary antibodies (Jackson Labs) were used at 1000× dilution. For immunofluorescence images, three independent fields at 20× magnification from six sections were imaged and CD206+ and CD68+ cells counted using imageJ.

At 21 days post-ischemia, after the last color laser Doppler analysis, mice were terminally anesthetized and the abdominal aorta was cannulated to perfuse the hindlimbs with 5 mM EDTA in PBS followed by 10% buffered formalin. The ischemic adductor muscle was harvested, stored overnight at room temperature (RT) in 4% paraformaldehyde (PFA), washed in PBS, and incubated in 30% sucrose for 24 hr before being embedded in optimal cutting temperature compound (OCT).
For analysis of capillary densities, adductor muscle sections were stained with isolectin B4 (Molecular Probes; I21411; 1:100) to identify the ECs and wheat germ agglutinin (Thermo Fisher Scientific; W32466) to stain muscle fiber. Slides were observed under a fluorescence microscope (Zeiss Z1 fluorescent microscope). 10 high-power fields were captured (×200), and the number of capillaries and muscle fibers per field were counted. Capillary density was expressed in two ways: (1) capillaries per mm² of transverse muscle section and (2) capillary number to myofiber number ratio. Capillary density quantification was conducted by two investigators blinded to the treatment groups.

The adhesion assay was performed to detect the adhesive ability of the mHSCs and hMSCs mediated by NAT-SDF-1α or CBD-SDF-1α. Briefly, a chemokine-modified collagen gel was prepared as the binding substrate in a 24-well plate. The mHSCs and hMSCs were seeded into each well (2 × 10⁴/well), and the medium was discarded after incubation at 37 °C for 1 hour. After gentle washing with PBS, the retained cells were fixed with 4% paraformaldehyde, and mHSCs were co-stained with anti-CD117 and DAPI. For MSCs, due to no unique marker, the retained cells were stained with DAPI only. Six fields of view were randomly imaged using a Zeiss Z1 fluorescent microscope, and the Hoechst-positive cells were counted.