Col-F Collagen Binding Reagent

1. When ready to use, reconstitute the 0.5 mg Col-F vial by adding 100 µL DMSO (this creates a stock at 6.8 mM). 2. Optional: Incubate samples with blocking buffer for 60 minutes. 3. Add Col-F to the media/buffer for each appropriate sample. 4. Incubate the samples with the dye solutions for 5-60 minutes at 37°C. 5. Wash samples twice with PBS (allow samples to incubate at room temp for 5-10 minutes in PBS during each wash step). 6. Once wash steps are complete, mount a coverslip on each slide. Samples are now ready to visualize with a fluorescence microscope capable of excitation at 490 nm, and emission at 515-520 nm.

Collagens and elastins are primarily synthesized by fibroblasts. These molecules are principal components of extracellular matrices. Once outside the cell, collagen assembles into fibrils and fibers that provide mechanical strength to tissues. Elastin is secreted by cells, and also forms fibers which crosslink to create a flexible network of fibers and sheets. Col-F is a low molecular weight fluorescent probe that exhibits affinity for collagen and elastin. Col-F easily penetrates between cells and into tissues where it can then bind to collagen and elastin fibers via hydrogen bonds and noncovalent hydrophobic interactions. Although Col-F has shown binding affinity for collagen and elastin, because the labeling is non-specific in nature, other tissue types may also be labeled. Analyze samples using a fluorescence microscope.

Q: 1. Can the stain retain for up to 48 hours without fixing the tissue after washing away excess Col-F? 2. Do you have the same dye for RED color (not green fluorescent)? A: We recommend that this product be used on fresh or frozen tissue samples, but not on formalinfixed paraffin-embedded samples. We have not gotten very good results when working with fixed and embedded tissues. Our product data sheet further describes our recommendations. In terms of Col-F staining for longer periods, such as up to 48 hours, we have not evaluated this experimentally However, we would expect the staining to be retained so long as the tissue is protected from light and tissue itself is remains stable and has not begun to break down. We do not currently have a product available in red fluorescence, though we may look at adding it in the future.