α sma ab5694

To test the possibility of obtaining cell sheets made of more than one cell population, MS1 (endothelial cells) and NIH-GFP⁺ (fibroblasts) cells were seeded onto 3D-printed supports filled with both MC-Na005 and MC-PBS20 hydrogels. Cells were seeded in a 50:50 ratio (1.5 × 10⁵ cells/support) and cultivated for 48 h. Co-cultured cell sheets were then detached by lowering the temperature as described above (Section 2.6.2), collected, and stained with an anti-alpha smooth muscle actin antibody (α-SMA, ab5694, from Abcam, Cambridge, UK) specific for endothelial cell cytoskeleton. Lastly, detached co-cultured cell sheets were co-stained with DAPI and images were collected using a fluorescence microscope (Leica DM5500 B).

Liver specimens were fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 4 μm sections and placed on glass slides. To examine hepatic morphology and assess liver fibrosis, liver sections were deparaffinized, rehydrated, and stained via the usual method with standard hematoxylin and eosin staining (H&E) and Sirius red staining as previously described [16 (link),25 (link)]. For immunohistochemical staining, liver sections were deparaffinized, rehydrated, and then incubated for 10 min in 3% hydrogen peroxide to block endogenous peroxidase. Antigen retrieval was performed by heating in 10 mM sodium citrate buffer (pH 6.0). Sections were blocked in DAKO protein block (X9090; DAKO, Carpinteria, CA, USA) for 30 min and incubated with primary antibodies at 4 °C overnight. The following primary antibodies were used: Tβ4 (A9520; diluted 1:8000; Immundiagnostik AG, Bensheim, Germany) and α-Sma (ab5694; diluted 1:1000; Abcam, Cambridge, MA, USA). Polymer horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (K4003; DAKO, Carpinteria, CA, USA) was used as secondary antibody. 3,3′-Diaminobenzidine (DAB) (K3466; DAKO, Carpinteria, CA, USA) was employed for the detection procedure.