the subepicardial space, belonged to this lineage. Whereas most of these cells populated the infarcted area replacing the killed cardiomyocytes, immunofluorescent analysis showed that a small portion differentiated into myofibroblasts, endothelial cells, and cardiomyocytes. Materials and Methods Animals All experimental procedures complied with national and institutional guidelines. The Animal Welfare Committee (DEC) of the University of Amsterdam approved this study and registered it as DAE101301 ‘‘Analyse van de epicard-afkomstige cellen in de regeneratie respons na een hartinfarct’’. The Wt1Cre transgenic line contains an IRES-EGFP-Cre cassette 17 bp downstream of the translation stop site of the Wt1 gene in the BAC clone RP23- 266M16 (http://bacpac.chori.org). This BAC clone ranges from 2127 kbp to +11.5 kbp relative to the transcription start site of Wt1 [19]. The Wt1Cre mice were bred with the R26R line [20] to visualize the Wt1 lineage. All mice were bred on a FVB background (Harlan, Indianapolis, IN) and wild-type littermates served as controls. Surgical induction of a myocardial infarction A myocardial infarction (MI) was induced by ligation of the left anterior descending (LAD) coronary artery at 8–12 weeks of age (20 gram body weight) as previously described [21]. Briefly, mice were anesthetized through intraperitoneal injection of Fentanyl (0.07 mg/kg), Dormicum (7 mg/kg), Dex-Domitor (0.35 mg/kg) and atropine (0.05 mg/kg). The mice were orally intubated using a 20-gauge intravenous catheter, mechanically ventilated (Mini- Vent, Harvard Biosciences), and placed on a heating pad to maintain body temperature. A left thoracotomy was performed at the third intercostal space, where muscles were dissected. The LAD was permanently ligated using a 9-0 unabsorbable ethilon suture. After visual verification of anemia and akinesis of the apex and anterior-lateral wall to ensure coronary occlusion, the thorax was closed in layers. The anesthesia was antagonized using Antisedan (2.5 mg/kg) and Flumazenil (0.5 mg/kg). After detu- bation, mice were kept warm until fully recovered. At least three mice were sacrificed at 1, 2, 3 days, 1 and 2 weeks, and 1 and 3 months after MI. Four hours before sacrifice the mice were injected intraperitoneally with 50 mg/kg 59-Bromo-29-Deoxyur- idine (BrdU; Sigma) solution. After sacrifice the hearts were flushed with phosphate-buffered saline (PBS) and cleaved. One half was snap frozen in liquid nitrogen and the other half was fixed overnight in freshly dissolved 4%(w/v) paraformaldehyde (PFA) in PBS. The fixed hearts were processed, embedded in paraplast and sectioned at 8 mm for in situ hybridization and immunofluorescent analysis. Quantitative PCR analysis The frozen hearts were mounted and 20 mm thick sections were cut using a cryotome. Twenty sections were collected in an eppendorf tube and an additional one was mounted for histological staining to evaluate whether the sections contained an extensive part of the infarct. If no or limited infarcted tissue was found the sample was excluded from the analysis. Total RNA was isolated from the sections using the RNeasy mini kit (Qiagen) according to the protocol of the supplier. One mg total RNA was converted into cDNA using a anchored polyT primer and SuperScript II Reverse Transcriptase (Invitrogen) in a 20 ml reaction. The RT reaction was diluted to 100 ml. Two ml single strand cDNA was used in each 5 ml qPCR reaction containing Endogenous Epicardial Regenerative Response 10 pMol of the respective forward en reverse primer and LightCycler 480 SYBR Green I Master. The following primers were used: Gapdh-FW: TGTCAGCAATGCATCCTGCA, Gapdh-RV: CCGTTCAGCTCTGGGATGA, Wt1-FW: CA- GATGAACCTAGGAGCTACCTTAAA, Wt1-RV: TGCCCT- TCTGTCCATTTCA, Tbx18-FW: GTGGAGTCATACGCAT- TCTGGA, Tbx18-RV: GTGAGGATGTGTAGCAGGGACA, Snai1-FW: CTTGTGTCTGCACGACCTGT, Snai1-RV: CAG- GAGAATGGCTTCTCACC, Snai2-FW: TCGTCGGCAGCT- CCACTCCA, Snai2-RV: TGTCAGAGGAAGGCGGGGGAC, Raldh1-FW: TCGAGTCCCTGCCCCACTGG, Raldh1-RV: TCCACGTGGCAGATGACCTCCT, Raldh2-FW: ATGAGT- GGCACGACGCCGTC, and Raldh2-RV: GCTGGAAGGC- TGCACGAGCA. After an initial incubation of 5 min 95uC, the cDNA was amplified in 40 cycles of 10 sec 95uC, 20 sec 60uC, and 20 sec 72uC using a Roche Lightcycler in 384 plates. The starting concentration in each reaction was calculated using the program LinRegPCR version 12.9 [22]. The observed value of Gapdh was used to correct each sample for differences in input. In situ hybridization In situ hybridization was performed essentially as previously described. [23] Sections were deparaffinized, rehydrated in a graded ethanol series and incubated with 10 mg/ml proteinase K dissolved in PBS for 15 min at 37uC. The proteinase K activity was blocked by rinsing the sections in 0.2% glycine in PBST (PBS+0.05%Tween-20) for 5 min. After rinsing in PBS, the sections were postfixed for 10 min in 4% PFA and 0.2% glutaraldehyde in PBS, followed by rinsing in PBS. After prehybridization for at least 1 hr at 70uC in hybridization mix (50%formamide, 56SSC (206SSC: 3 M NaCl, 0.3 M tri-sodium citrate, pH4.5), 1% blocking solution (Roche), 5 mM EDTA, 0.1% 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfo- nate (Sigma; Steinheim, Germany), 0.5 mg/ml heparin (BD Biosciences; Erembodegem, Belgium), and 1 mg/ml yeast total RNA (Roche), a digoxigenin (DIG)-labeled probe was added to the hybridization mix in a final concentration of 1 ng/ml. Probes specific to cardiac Troponin I (cTnI), Raldh1, Raldh2, Wt1, Tbx18, Snai1, Periostin and Fstl1 were used. After overnight hybridization, the sections were rinsed with 26SSC, followed by two washes with 50% formamide, 26SSC, pH 4.5, at 65uC, and rinsing in TNT (0.1 M Tris-HCl, pH7.5, 0.15 M NaCl, 0.05% Tween-20) at room temperature. Subsequently, the sections were incubated for 1 hr in MABT-block (100 mM Maleic Acid, 150 mM NaCl, pH7.4, 0.05% Tween-20, 2% blocking solution), followed by 2 hours incubation in MABT-block containing 100 mU/ml alkaline phosphatase-conjugated anti-DIG Fab frag- ments (Roche catnr: 1093274). After rinsing in TNT and subsequently in NTM (100 mM Tris pH9.0 100 mM NaCl, 50 mM MgCl2), probe binding was visualized using nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate (Roche catnr: 1681451). Color development was stopped by rinsing in double-distilled water. The sections were dehydrated in a graded ethanol series, rinsed in xylene, and embedded in Entellan. Images were recorded using a Leica DFC320 camera mounted on an AxioPhot microscope (Zeiss). Immunofluorescent staining Immunofluorescent staining was essentially performed as described. [24] In short, sections were deparaffinized, rehydrated in a graded ethanol series, boiled for 5 minutes in antigen unmasking solution (H3300, Vector), and incubated in PBS+1% Triton-6100 for 15 min. The following primary antibodies were used: anti-Troponin I (TnI) (MilliPore; catnr MB1691; 1:500), PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e44692