Ki67 clone tec 3

Antibody stains were performed on paraffin sections. Tissues were fixed overnight in 4% paraformaldehyde (PFA), dehydrated by washing through an ethanol gradient series, washed in histoclear and embedded in wax. 4 µm sections were cut, treated with histoclear and rehydrated through an ethanol gradient series. Antigen retrieval was obtained by boiling the sections in citrate buffer (0.1 M sodium citrate pH6 and 0.05% Tween). Sections were incubated in PBS with 10% sheep serum and then incubated with primary and secondary antibodies in PBS with 1% sheep serum. Sections were counterstained with DAPI nuclear stain. The following antibodies were used for immunohistochemistry; SOX9 was a kind gift from Francis Poulat (CNRS, Montpellier, France, 1:2000), Ki67 (clone TEC-3, Dako, 1:20), E-Cadherin (BD Transduction Labs, 1:50). Secondary fluorescent antibodies were obtained from Molecular Probes and were used at a 1:500 dilution. Fluorescent images were visualized and collected on a Leica TCS-SP2 confocal microscope.

The infarcted area was determined in serial sections (5 μm) of the infarcted myocardium (10 sections per mouse) after staining with Gomori*s 1-step trichrome staining using Diskus software (Hilgers, Königswinter, Germany) and expressed as percentage of LV area. The number of neutrophils (naphthol-AS-D-chloroacetate esterase and anti-MPO, #RB-373-A; Thermo Scientific, Waltham, MA, USA) was determined 1 day after MI, whereas the endothelial cells (CD31, #M20; Santa Cruz, Santa Cruz, CA, USA) was determined in formalin-fixed serial sections (three per mouse, 200 μm apart) of the infarcted myocardium 4 weeks after MI. Positive-stained cells were numbered in six different fields from infarcted area per section and expressed as cells/mm². Blood vessels positive for CD31 were quantified and expressed as CD31/mm². Apoptotic (TUNEL, TMRred; Roche, Mannheim, Germany) and proliferating (Ki67, clone TEC-3; DAKO, Hamburg, Germany) cells were stained, counted and expressed as per cent from total cells (DAPI staining). Total Akt (Akt1/2 (N-19), sc-1619; Santa Cruz) and phosphorylated-Akt (phospho-Akt (Ser) (193H12), #4058S; Cell Signaling Technologies, Beverly, MA, USA) were stained using Dylight 488–conjugated secondary antibodies (Thermo Scientific).