Collagen IV fluorescent labelling

5 mg of type IV human placenta collagen (Sigma-Aldrich, C7521) was reconstituted in 5 mL of phosphate buffer (PBS). The sample was split into 10 0.5 mL aliquots and stored at −20 °C. 1 mg of NHS activated AlexaFluor480 green fluorescent dye (Invitrogen) was dissolved in 200 µL dimethylformamide. Sample was split into 20 10 µL aliquots and stored at −20 °C. 28 µL of AlexaFluor480 (5 mg/mL) was added in a solution of 500 µL of collagen (1 mg/mL). Reactions were allowed to progress on a tube roller for 1 h 15 min at room temperature. Unreacted dye was removed by centrifugal filtration at 3000 RCF, using 10000 MWCO spin filters. Protein was washed three times with water. Dye conjugated collagen was collected from the filter surface after dismantling the filter apparatus by immersion in 1 mL PBS for 5 minutes under agitation (tube roller). Green-labelled protein transferred on HH masks and PAm hydrogels was detected using a fluorescence confocal microscope (Zeiss Axiovert 200).

Fluorescent micrographs of micro patterned FITC-labelled protein were analyzed by using ImageJ software (available at http://rsbweb.nih.gov/ij/download.html) to extract the intensity profiles.