Prepare the ORO solution by dissolving 0.5 grams of ORO powder in 100 milliliters of isopropanol to generate a stock solution. Prepare the working solution for all muscles by combining the ORO stock solution and deionized water at a 60 to 40 ratio. Cover the working solution for 10 minutes to allow the particulate to settle.
Filter it through a 40 micrometer mesh, followed by a 0.22 micrometer syringe filter. Next, remove the formaldehyde solution from the decellularized muscles in the well plate and wash the muscles three times with an equal volume of PBS. Then, replace the PBS with an equal volume of 60%isopropanol solution and incubate it on the rocking shaker for five minutes.
Once done, replace the 60%isopropanol solution with ORO working solution before 10 minutes of incubation on the rocking shaker. Next, replace the ORO working solution with an equal volume of 1%SDS and inspect the SDS solution periodically. Once the solution becomes noticeably pink, replace the SDS solution with fresh 1%SDS.
When the solution remains clear for 24 hours, replace the 1%SDS with PBS. Under a stereo microscope at four x magnification, remove any obvious debris or particulate stuck to the outside of the stained muscle. If the staining is satisfactory, showing the bright red spheres floating in a transparent matrix, acquire the staining images using a camera attached to the stereo microscope.
Properly decellularized muscles were white and semi-transparent. When decellularized muscles were stained with ORO to visualized intramuscular adipose tissue, or IMAT, IMAT lipid droplets were apparent within the clear muscle structures as red spheres. Healthy mouse hind limb muscles showed a little natural IMAT evidenced by little to no red or positive lipid as compared to the hind limb muscles injected with cardio toxin or glycerol.
Incomplete decellularization was identified immediately following initial SDS treatment or after the washout of the ORO staining as semi opaque light pink fibers. Incomplete ORO clearance was identified following ORO washout as a pink or red uniform background rather than distinct fiber lines.
