2.3.2. SEM and Fluorescent Microscopy of Matrices with HUVEC
This protocol is extracted from research article:
Assessment of Electrospun Pellethane-Based Scaffolds for Vascular Tissue Engineering
Materials (Basel), Jul 1, 2021; DOI: 10.3390/ma14133678

The matrices with HUVEC after 48 h incubation were washed twice with phosphate buffer to remove non-adherent cells and fixed in 2% formaldehyde in a phosphate buffer for 30 min. Graded ethanol series (50%, 70%, 90%, and 100%) were used for sample dehydration before incubation in a mixture of hexamethyldisilazane (HMDS) with ethanol (1:1), and then 100% HMDS in order to prepare 3D matrices with cells for study by a EVO 10 (Carl Zeiss AG, Jena, Germany) scanning electron microscope.

HUVEC cells cultivated at 3D matrices during 48 h were fixed in 2% formaldehyde in a phosphate buffer and stained with Hoechst 33342 (Invitrogen, USA) and Phalloidin-TRITC (Sigma, USA) for 20 min at 37 °C for subsequent fluorescent microscopy. For this study, we used a confocal laser scanning microscope LSM 780 NLO (Carl Zeiss, Jena, Germany) at 405 nm to detect cell nuclei and at 543 nm to detect actin microfilaments with ZEN 2 software (Carl Zeiss Inc., München, Germany).

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