Ma5 11358

EpC cells were viable after unfreezing and were seeded on coverslips in a 24-well plate (5 × 10⁴ cells) for 7 days. Cells were PFA (Synth) fixed for 20 minutes. Coverslips were washed in PBS 1% + 0.5% Tween-20 (Synth) solution and incubated with primary antibodies diluted in 2% PBS + BSA: E-Cadherin (24E10, Cell Signaling, 1:200), N-Cadherin (13A9, Cell Signaling, 1:200), Vimentin (GTX35160, GeneTex, 1:100), Cytokeratin 18 (RGE53, Novus, 1:200), β2 Tubulin (sc-47751, Santa Cruz Biotechnology, 1:200), VEGF (ac12013315, Bioss), CD44 (#GTX80086,Genetex,1:100), TGF-β (#SC-146, Santa Cruz Biotechnology, 1:100), CD31 (#ab32457, Abcam, 1;100), PCNA (ma5-11358, Invitrogen, 1:100) and hyaluronic acid (#c41975, LS Bio, 1:100) for 1 hour at 37°C. Then, Alexafluor 488/594 secondary antibodies (#A30008/#A-11094, Thermo Fisher) were added at concentration 1:200 for 1 hour at 4°C. Recellularized scaffolds and the coverslips were carefully washed, followed by DAPi (Sigma-Aldrich) incubation for 10 minutes for nuclei stain. The samples were analyzed in Confocal Microscope – Olympus Fluo View 1000 (CADI-FMVZ).

In vivo implanted samples in the subcutaneous tissue of mice were collected and analyzed by immunohistochemistry using the Dako EnVisionTM FLEX Detection System, High pH Link (#K8000) according to the manufacturer’s instructions. The slides were washed in a buffer solution and the sections surrounded with a hydrophobic pen. For ECM analysis, the primary antibodies diluted in PBS + 2% bovine serum albumin (BSA, #A1310-05, LGC Biotechnology) were used: PCNA (ma5-11358, Invitrogen, 1:100), CD68 (ab9555, abcam, 1:200), CD4 (PA0427, Leica, 1:200), CD8 (PA0183, Leica, 1:200). Following incubation time, the slides were washed, and HRP solution were incubated for 30 minutes in a humid chamber. Subsequently, the sections revels was performed using a reaction with diaminobenzidine (DAB, 30 μl per section: 1 ml of substrate + 1 drop of chromogen). Slides were counterstained with Harris Hematoxylin for 40 seconds, washed with H₂O distilled and rehydrated. The samples were photodocumented in a Nikon Eclipse 80i microscope.