F ab 2 donkey anti rabbit igg pe

For characterization of BMSCs, BMSCs were analysed by fluorescence‐activated cell sorting (FACS). Cells in the fourth passage were incubated in antimouse/rat CD29 FITC (1:200, eBioscience), anti‐rat CD44H PE (1:300, eBioscience), anti‐rat CD45 APC (1:400, eBioscience), antimouse/rat CD90.1 PerCP‐cyanine5.5 (1:400, eBioscience) or CD34 antibody (ICO115) FITC (1:10 dilution, Santa Cruz) at concentrations specified by the manufacturer. Corresponding isotype identical antibodies served as controls, and the concentrations of the isotype identical antibodies were the same as the labelled antibodies.
Regarding CXCR4 expression, cells were stained with anti‐CXCR4 antibody (1:50, Abcam) or IgG isotype control, and the secondary antibody was F (ab’) 2 donkey anti‐rabbit IgG PE (1:50, eBioscience). To study the effects of simvastatin on the surface expression of CXCR4, 1 μmol/l simvastatin was added into the culture medium 48 h before harvest. To determine the effect of miR‐9, cells were harvested 48 h after transient transfection.

Neutrophils were washed twice in PBS after in vitro stimulation. Fc receptors were blocked for 20 min at RT with anti-mouse or anti-human CD16/32 antibody (eBiosciences 16-0161-81) followed by incubation with FITC tagged anti-Ly6G (clone 1A8, Biolegend, 127613, 0.5 μg added to 1 × 10⁶ murine neutrophils in 100 μl) and anti-P2X₇ receptor antibodies as described above. F(ab')2 donkey anti-rabbit IgG-PE (eBiosciences) was used as secondary antibody for P2X₇. Following two washes in FACS Buffer (1% FBS in PBS), cells were fixed in 0.5% PFA for analysis by flow cytometry using an Accuri C6 Flow cytometer (Becton Dickinson).

Confocal imaging was performed on NF and HF arteries after ligation as previously reported [45 (link)]. Arterial segments were stained overnight (4 °C) with anti-mouse CD45-PE (clone 30-F11, rat IgG2bκ isotype, 1:200, BioLegend, San Diego, CA, USA), anti-mouse Ly-6G (Gr-1)-PE (clone RB6-8C5, rat IgG2bκ isotype, 1:200, eBioscience, San Diego, CA, USA), anti-mouse F4/80-PE (clone BM8, rat IgG2aκ isotype, 1:200, eBioscience) or with anti-cleaved caspase-3 Ab2 (1:100, Calbiochem, San Diego, CA, USA) and with F(ab’)2 donkey anti-rabbit IgG PE (1:100, 2 h, 20 °C, eBioscience). Samples were then washed with PBS three times for 5 min and mounted with Mowiol. Nuclei were stained with DAPI (Invitrogen, Waltham, MA, USA) 11 µg/mL. All dilutions were made in PBS-BSA 5%. Pictures were taken with a Nikon Eclipse TE2000S confocal microscope and analyzed with the MetaMorph^® software (San Jose, CA, USA).