Acidic Collagen Lab Report

Decent Essays

Acidic collagen type I (CosmoBio) at 5 mg/mL concentration and pH 3 was used. Specified volumes of collagen solution were dispensed into plastic molds maintained at 4°C. In a closed chamber, ammonium hydroxide maintained at 25°C was allowed to interact with degassed collagen solution. Collagen was exposed to ammonia vapors in desiccation chamber at room temperature for 3 min, 30 min and 2 h. Collagen gels were then removed from ammonia chamber and incubated in 5°C vitrification chamber for 90 minutes, followed by a 30-minute incubation (gelation) at 37°C. Samples were vitrified in a 5°C vitrification chamber overnight followed by a 37 °C and 40% RH for 3 days. The biomaterials were then rehydrated in 20 mM HEPES for 24 hours.
6.2.2. Second Harmonic Generation Microscopy
To observe fibrogenesis of AMC vitrigels, two-photon microscopy was performed using a Zeiss LSM-710 microscope (Carl Zeiss, Jena, Germany. Illumination was performed at a wavelength of 790 nm. Collagen fibrils were detected by its second-harmonic generation (SHG) signal below 480 nm. Images were recorded as z-stacks overarching the whole membrane with 1 µm Z-spacing using Zen software 2009 (Carl Zeiss).
6.2.3. Light Transmission
AMC vitrigels were cast in microplate wells, vitrified, rehydrated and measured using a standard multiplate reader Synergy 2 (Biotek). Area scan measurements were made at over the visible spectrum at 50 nm intervals. Data was compiled and analyzed in Excel.
6.2.4. Scanning Electron

Get Access