Vascular endothelial growth factor (vegf)

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).

The cryptorchid tissue and healthy testicular tissue of Erhualian pigs fixed with 4% paraformaldehyde solution at room temperature could be preserved for a long time. Tissue blocks used for the test were embedded in paraffin, sliced, dried, and preserved at 4 °C.
To detect the positive immunohistochemical reactions of VEGF/EGFR and P53/NF-κB, all the prophase experimental steps were completed in strict accordance with the immunohistochemical procedure. They were treated with 3% deionized H₂O₂ for 15 to 20 min and sealed with goat serum for 15 to 20 min, added to monoclonal antibody VEGF (goat anti-mouse, 1:300, Bioss, Beijing, China), polyclonal antibody EGFR (goat anti-rabbit, 1:400, CST, USA), monoclonal antibody P53 (sheep against mouse, 1:500, Abcam, Boston, MA, USA), and polyclonal antibody NF-κB (goat anti-rabbit, 1:400, CST, USA), and incubated overnight. After washing, the corresponding secondary antibody was added, and tissues were incubated (Goat Anti-Mouse IgG (H+L)/HRP, bs-40296, bioss, diluted at 1:300; Goat Anti-Rabbit IgG (H+L)/HRP, bs-40295G, bioss, 1:300 dilution).

For the IHC, the primary antibodies used for immunochemistry were as follows: VEGF (Bioss, bs-1665R) and Nestin (Abcam, ab7659). Secondary antibodies were all purchased from Boster in China. In brief, the slides were deparaffinized and rehydrated by gradient elution using xylene and ethanol, followed by incubation in 3% H₂O₂ to suppress endogenous peroxidase activity. For antigen retrieval, the slides were incubated in hyaluronidase (Sigma-Aldrich, St Louis, MO, USA) for 1 h at 37 °C. After the sealing of 5% BSA, the specimens were incubated with primary antibody diluted 1:100 at 4 °C overnight. Then sections were then rinsed in PBS and incubated with biotinylated secondary antibody for 20 min at room temperature. SABC kit purchased from Boster (Wuhan, China) was used for the staining process. 3,3′-Diaminobenzidine (DAB) was used as a color developing agent, and then slides were counterstained with hematoxylin. Finally, slides were mounted with Permount TM Mounting Medium and observed by the stereomicroscope (Carl Zeiss, Stemi 508, Germany) and the microscope (Nikon Eclipse 80i, Japan). Pictures were modified by PhotoshopCS6 (Adobe, CA, USA). Negative controls were incubated with normal anti-rabbit or anti-mouse IgG instead of the primary antibodies.