*4.11. Immunohistochemistry*

CAM sections were deparaffinized and rehydrated in an MICROM HMS740 automatic stainer (MICROM, Walldorf, Germany). Immunohistochemical analysis was performed using a streptavidin–biotin peroxidase complex system. Briefly, after rehydration, slides were subjected to heat-induced antigen-retrieval with 10 mM citrate buffer at pH = 6 for 2 min at 98 ◦C. The slides were washed with PBS and then incubated with 3% hydrogen peroxide for 10 min to inactivate endogenous peroxidases. Another washing step was performed, and nonspecific binding was blocked with a 2.5% (*v/v*) horse serum (Vector Labs, Newark, CA, USA) for 30 min, before overnight incubation at 4 ◦C with mouse anti-human CD31 (1:30) (Dako, Cambridge, UK). Sections were washed with 0.1% tween in PBS and incubated with a secondary biotinylated antibody (Vector Labs, Newark, CA, USA) for 20 min. After thoroughly washed with 0.1% tween in PBS, samples were incubated with streptavidin-HRP (Vector Labs, Newark, CA, USA) for 20 min, followed by 3,3diaminobenzidine (DAB) incubation (Vector Labs, Newark, CA, USA). Finally, all sections were counterstained with Mayer's hematoxylin, dehydrated and mounted with resinous mounting medium Entellan®(Merck, Darmstadt, Germany).

### *4.12. In Situ Hybridization*

The presence of human cells within the implantation area was assessed using a human-specific DNA probe, according to the respective detection system BIO-AP REM-BRANDT ®Universal DISH & Detection kit (PanPath, Budel, The Netherlands). Briefly, after deparaffinization, proteolytic digestion was performed using a pepsin-HCL solution for 30 min at 37 ◦C, followed by dehydration in graded ethanol series. Sections were air-dried, and 1 drop of the probe was applied and covered with a coverslip. Samples were incubated at 95 ◦C for 5 min for DNA denaturation and then for 16 h at 37 ◦C in a moisturized environment for hybridization to occur. Samples were then washed in Tris-buffered saline (TBS) and incubated for 10 min with the stringency wash buffer. After rinsing with TBS, the detection was performed, and color was permitted to develop for 5 min at 37 ◦C in the dark. Samples were washed with water, counterstained with nuclear fast red, and observed under a Leica DM750 microscope (Leica, Wetzlar, Germany).
