Rabbit anti collagen 1

Immunofluorescence studies were carried out to identify the presence of collagen type I and IV on the surface of the dip-coated and LS films. First of all, films were incubated (1h, RT) with 3% bovine serum albumin (BSA, Sigma-Aldrich, USA), and subsequently with the primary antibodies, i.e. mouse anticollagen IV (Millipore, USA) and rabbit anti-collagen I (Millipore, USA), at a 1:200 ratio in 0.5% BSA-DPBS (overnight, 4 °C). After washing with DPBS (3x), films were incubated (1h, RT) with the secondary antibodies: donkey anti-mouse Alexa Fluor 488 (Molecular Probes, Life Technologies, USA) and goat anti-rabbit Alexa Fluor 568, both diluted to ratio of 1:800 in 0.5% BSA in DPBS. Before imaging, films were washed again with DPBS (3x). Secondary antibody controls were similarly prepared but excluding the incubation step with the primary controls. Collagen distribution on sample surface was imaged using a Zeiss LSM780 Laser Scanning Confocal Microscope (Carl Zeiss, Germany) and a Plan-Apochromat 63x/1.2 (water) objective (Carl Zeiss, Germany). Samples were further compared to the uncoated controls.