A morphological study of the ECM skeletons was realized with an advanced environmental scanning electron microscope (A-ESEM) [27 (link)], in combination with the low temperature method (LTM) [28 (link),29 (link)]. Briefly, tissue samples were fixed in buffered 4% formaldehyde overnight. Small parts of ECM skeletons and pancreases (up to 5 × 5 mm) were cut for samples, washed 2 times in distilled water, and placed on a cooled specimen holder (Peltier stage). The LTM procedure began with cooling to 0 °C, then the pumping process in the specimen chamber was started as sample cooling continued to −22.5 °C. All experiments were carried out in our customized QUANTA 650 Field Emission Gun SEM (Thermo Fisher Scientific, Waltham, MA, USA), under the following constant operating conditions: 150 Pa water vapor pressure, 10 kV beam accelerating voltage, 45 pA beam current, and 7.5 mm working distance. Sample topography was imaged with an ionization secondary electron detector, equipped with an electrostatic separator. The material contrast of the iron nanoparticles was studied with a scintillation detector to detect the backscattered electrons [30 (link)]. Macrographic images were taken of the ECM skeletons, and the whole image was composed by merging micrographs with Maps software (Thermo Fisher Scientific, Waltham, MA, USA).
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