Cryosectioning and Histological Staining

The cell sheets were fixed in 4% paraformaldehyde/PBS, cryoprotected with sucrose, embedded in tissue freezing medium (Jung, Leica, Nussloch, Baden-Württemberg, Germany) and cryosectioned at 10 μm (Microm HM500 OM, Fisher, Walldorf, Baden-Württemberg, Germany). Sections were stored at −20 °C until use. Before staining, sections were equilibrated to room temperature and hydrated with PBS (3 × 5 min) and rinsed with deionized water. For Haematoxylin and Eosin (H&E), sections were placed in a ready-to-use Hematoxylin solution (Carl Roth, Karlsruhe, Baden-Württemberg, Germany) for 10 min, rinsed with deionized water for 5 min and washed in tap water for 7 min. Next, sections were immersed in a ready-to-use Eosin solution (Carl Roth, Karlsruhe, Baden-Württemberg, Germany) for 10 min, rinsed with distilled water for 30 s, and mounted. Alexa Flour 546-labelled phalloidin (Invitrogen, Karlsruhe, Baden-Württemberg, Germany) was applied according to the manufacturer’s recommendation, as described previously [6 (link)]. DAPI was used for nuclear counterstaining. Cell sheets were imaged with the AxioCamMRm camera on the AxiovertS100 microscope (Carl Zeiss, Jena, Thüringen, Germany). For quantification of the total actin amount, fluorescence signals were quantified using the algorithm described above.