Cytokeratin pan ab

The staining of Col I (anti-collagen I antibody, 1:2000, Abcam, Cambridge, UK) and αSMA (anti-actin α-smooth muscle, 1:400, Sigma) was evaluated after 7 and 14 d in SC monocultures on scPLCL and scPLCL_A2P. Additionally, in cocultures of ECs and SCs on scPLCL_A2P, the staining of pancytokeratin (AE1/AE3, 1:250, Cytokeratin Pan Ab, Thermo Fisher Scientific) and actin cytoskeleton organization (phalloidin-tetramethylrhodamine B isothiocyanate, 1:500, Sigma-Aldrich) was evaluated after 7 and 14 d of cell culturing.
The samples were fixed with 0.2% Triton X-100 (Sigma-Aldrich) in 4% PFA (Sigma-Aldrich) and incubated overnight in the abovementioned primary antibody dilutions. The following day, the SC monocultures were incubated in secondary antibody dilutions (1:400, goat anti-mouse IgG1 or 1:300, goat anti-mouse IgG (H + L), Alexa-fluor 488, green fluorescence, Invitrogen). The EC and SC cocultures were incubated in a mixture of secondary antibody (1:400, goat anti-mouse IgG (H + L), Alexa-fluor 488, green fluorescence, Invitrogen) and phalloidin. Finally, the cell nuclei were stained with DAPI (1:200, blue fluorescence, Sigma-Aldrich), and the samples were imaged with a fluorescence microscope (Olympus).

The immunostaining was used to evaluate the expression of cytokeratins (AE1/AE3 pancytokeratin, 1 : 250, Cytokeratin Pan Ab, Thermo Scientific) and actin cytoskeleton organization (phalloidin-tetramethylrhodamine B isothiocyanate, 1 : 500, Sigma-Aldrich) in co-cultures of ECs and SCs at 7 and 14 day time point. Briefly, the samples were fixed with 4% paraformaldehyde (Sigma-Aldrich) and incubated overnight in pancytokeratin primary antibody dilutions. The next day, the samples were incubated in a mixture of secondary antibody (1 : 800 Alexa-488 donkey, green fluorescence) and phalloidin. Finally, the cell nuclei were stained with DAPI (1 : 2000, blue fluorescence, Sigma-Aldrich), and the cells were imaged with a fluorescence microscope (Olympus).