Fei quanta 250 feg scanning electron microscope

Electrospun samples seeded
with neuro2a cells were fixed in 2.5% glutaraldehyde solution buffered
in a 0.1 M sodium cacodylate solution, pH 7.2. After the fixation
process, samples were washed with 0.1 M sodium cacodylate buffer,
pH 7.2, and submitted to a process of metallic impregnation. To make
this, samples were incubated in 2% osmium tetroxide in 0.1 M sodium
cacodylate buffer, pH 7.2 for 2 h, washed with 0.1 M sodium cacodylate
buffer pH 7.2 three times during 15 min, and incubated in 1% tannic
acid water solution for 45 min, followed by two washes in distilled
water (10 min each).
After metallic impregnation, samples were
dehydrated gradually in 50–70–90% ethanol (twice, 30
min each) and 100% (three times, 30 min each), and then samples were
submitted to a drying process in a critical point chamber (Balzers
CPD 030, Lichtenstein) using CO₂. Samples were then coated
with a thin layer of 20–30 nm thickness of gold (Sputtering,
Leica Microsystems, Germany) and scanned on a FEI Quanta 250 FEG scanning
electron microscope (ThermoFisher, U.S.A.).

Rat tail type I telocollagen was diluted to a final concentration of 1.5 mg/ml and supplemented with different PGs as descried above for the spectrometric (turbidity) assay. It was polymerized on 8 mm coverslips for 25 min. The bovine atelocollagen gel sample was made similarly. The collagen gels were fixed with 2.5% glutaraldehyde in cacodylate buffer overnight at 4 °C. The samples were further processed by the Cell and Developmental Biology Microscopy Core (University of Pennsylvania, Philadelphia, PA, USA). Briefly, samples were dehydrated with a graded series of ethanol washes (50, 75, 90, 95, 100%) and incubated with 50% hexamethyldisilazane (HDMS) for 30 min. Samples were then incubated with 100% HDMS three times and air dried before mounting on stubs. Samples were imaged on a FEI Quanta 250 FEG scanning electron microscope (Thermo Scientific). Bovine atelocollagen was prepared and studied in the same manner. 5 SEM images were taken per each gel at 5 random locations with 10,000× magnitude. 5 randomly-cropped figures (384 × 256 pixels) from each SEM image were analyzed using DiameterJ, an imageJ plugin, which was used to quantify fiber diameter and network porosity (porosity is the area of pores over the total area of the figure)^{64 (link)}.