Immunostaining and Trichrome Staining of Cell and Tissue Samples

Cells in lrECM gel were smeared on slides, dried briefly, and fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Immunostaining was performed as previous described [32 (link)]. Stained samples were imaged with a Nikon upright epifluorescence microscope or a confocal system comprised of an Olympus IX81 microscope.
Xenograft tumor sections were de-paraffined and hydrated from xylene, 100% ethanol, 95% ethanol, 85% ethanol and 70% ethanol to distilled water. For Masson’s trichrome staining, slides were re-fixed with Bouin’s solution at 60°C for 60 minutes. Slides were washed in running tap water for 5 minutes and stained in Weigert’s working hematoxyin for 10 minutes. Then they were washed in running tap water for 5 minutes and stained in Biebrich scarlet-acid fuchsin solution for 5 minutes. Slides were rinsed in distilled water and differentiated in phosphomolybdic-phosphotungstic acid solution for 10 minutes, transferred to aniline blue solution and stain for 5 minutes. Slides were rinsed in distilled water and images were taken with a Nikon microscope. The percentage of collagen was quantified by calculating the ratio of blue staining (collagen) area in the total area of the tumor section using Imagescope analysis software [33 (link)].