Figure 2

The Scar-in-the-Jar system combines enhanced collagen deposition with optical analysis for in situ quantitation. (A) Cell layers were pepsin digested, resolved by sodium dodecyl sulphate - polyacrylamide and silver stained. In comparison with fibroplasia models (FP1: Ref [61], FP2: Ref [45]), macromolecular crowding increased matrix formation including stronger lysyl oxidase-mediated cross-linking in both deposition modes (rapid: dextran sulphate [DxS]; accelerated: Ficoll cocktail [Fc]), within a shorter time frame. Note: the presence of collagen V in FP and the accelerated deposition mode and its absence in the rapid deposition mode. Collagen V is usually absent from fibrotic tissue; hence, the extracellular matrix obtained in the rapid deposition mode will probably be more similar to a fibrotic matrix. (B) Cell layers were immunostained for collagen I and fibronectin. Cell nuclei were stained with 4', 6-diamidino-2-phenylindoldilactate (DAPI). The rapid deposition mode (negatively charged, DxS) produces granular collagen I and fibronectin within 2 days, and the accelerated mode (neutral, Fc) produces collagen I with a reticular deposition pattern within 6 days. Therefore, the amount, velocity and morphology of deposited collagen can be manipulated depending on the macromolecules used. (C) Optical analysis of deposited collagen I using a 2× objective, eliminated corner auto-fluorescence in the four corner fields with triangular masks to conceal these regions during quantitation. (D) Cytometry and quantitation of the area of deposited collagen I in a 24-well multiplate format enabled identification of antifibrotic substances that perturb the collagen biosynthesis pathway resulting in a net reduction of deposited collagen I. (i) DAPI-stained nuclei at 20× total magnification in monochrome pseudocolour, 600× magnification (inset). (ii) Red scored nuclei by Count Nuclei module for cytometry. (iii) Immunostained deposited collagen I. (iv) Regions with fluorescent pixel intensity above a selected value based on controls are demarcated by the software in green for quantitation of deposited collagen I area at 100× magnification. This figure is reproduced with permission (Ref [59]).