Journal: Current protocols in cell biology

Article Title: Preparation of extracellular matrices produced by cultured and primary fibroblasts

doi: 10.1002/cpcb.2

Figure Lengend Snippet: Unextracted fibroblast-derived 3-D matrix phenotypes. (A-F) Representative confocal indirect immunofluorescent reconstructed images, corresponding to assorted unextracted human pancreatic primary fibroblasts-derived 3-D matrix cultures. Normal vs. desmoplastic (i.e., myofibroblastic activated) monochromatic matrix (i.e., fibronectin; A–B) and α-SMA (C–D) images are shown while corresponding nuclei images are in the inserts. The same overlaied images are shown in panels E and F; green fibronectin, red α-SMA and blue nuclei. G and H panels depict the OrientationJ software’s visual output obtained from A and B. Colored bars to the right represent the angles revealed by the software, while the corresponding mode angle color is shown for each image. I and J are the resultant Hue-corrected images displaying mode angle in Cyan representing normalized image/color (i.e., 0°). Note how color variations are diminished in the desmoplastic example where the majority of fibers are parallel oriented. Bars represent 50 μm. Graphs in K constitute numeral outputs from G and H while L corresponds to the normalized distributions (analogous to I and J).

Article Snippet: Dulbecco’s phosphate-buffered saline (DPBS + ; APPENDIX 2A ) DPBS + with Tween-20 (DPBS-T; see recipe) Fixing/Permeabilization solution (see recipe) Fixing solution (see recipe) Fibroblast-derived 3-D matrix produced onto 12-mm no. 1.0 coverslips (see Basic Protocol) 100% Donkey serum stock (Jackson ImmunoResearch Laboratories) Odyssey® Blocking Buffer (LI-COR Biosciences, P/N 927-40000) with 1% donkey serum (see recipe) Primary antibody cocktail: anti-α-Smooth Muscle Actin (α-SMA) mouse antibody [1:300] (Sigma-Aldrich, catalog number: A2547), anti-fibronectin rabbit antibody [1:200] (for murine samples use Abcam, catalog number: ab23750; for human samples use Sigma, catalog number: F3648,).

Techniques: Derivative Assay, Software