239 Fibroblast remodeling of extracellular matrix is directed by the fibrotic nature of the threedimensional microenvironment Picrosirius Red Staining for visualization of collagens PicroSirius Red (PSR) solution was prepared using 0.5 g Sirius red F3B (Sigma) in 500 mL saturated aqueous picric acid solution. Deparaffinized slides were washed with distilled water and then incubated with the PSR solution for an hour. Afterwards, the slides were washed with acidified water (5 mL glacial acetic acid (Merck) in 11 mL distilled water) twice and dehydrated through 75%, 96%, 100% ethanol (Fresenius Kabi) and xylene (Klinipath BV) solutions. After airdrying the slides, they were mounted using a non-aqueous mounting medium and kept in dark until imaging. Alcian Blue Staining for visualization of glycosaminoglycans Alcian blue solution was prepared using 1 g Alcian blue (Sigma-Aldrich) powder in 100 mL 3% acetic acid solution. Nuclear fast red solution was prepared using 0.1 g nuclear fast red powder in 100 mL distilled water with 5 g Al2(SO4)3 (Sigma-Aldrich). Deparaffinized slides were washed with distilled water and then incubated with the Alcian blue solution for 30 minutes at room temperature. Afterwards, the slides were washed with running tap water for 2 minutes and rinsed in distilled water. Counterstaining was performed through incubation with nuclear fast red solution for 5 minutes and the slides were washed in running tap water for 1 minute. The slides then were dehydrated through 75%, 96%, 100% ethanol (Fresenius Kabi) and xylene (Klinipath BV) solutions. After airdrying the slides, they were mounted using a nonaqueous mounting medium and kept in dark until imaging. Imaging Immunohistochemical staining results were imaged using a Hamamatsu scanner (Hamamatsu Photonics K.K., Herrsching, Germany) at 40X magnification. Fluorescent microscopy images of the PSR stained hydrogels were captured with Leica SP8X white light laser confocal microscope (Leica, Wetzlar, Germany) with UV-vis absorption (λex) 561 nm and emission (λem) 566 / 670 nm using 63x/1.40 Oil immersion lens with a digital zoom 2X. Image Analysis Analysis of Light Microscopy Images Specific image areas with stained gel were extracted into TIF (LZW) files using Aperio ImageScope V12.4.0.5043 (Leica Biosystems, Amsterdam, Netherlands). These TIF files were opened in Adobe Photoshop CS6 Extended (San Jose, California, USA) and artifacts, such as obvious background staining, hairs or other contaminations and folded areas of gels, were removed. Ten areas from ten distinct regions per image (1 image per gel) were randomly selected and saved as separate image files to enable 9
RkJQdWJsaXNoZXIy MTk4NDMw