To begin, open the software compatible with the load frame instrument, and select an existing control file for analysis. Turn on the actuator. In the calibration menu, tear the force reading of the load cell and set the displacement of the actuator to zero.
After ensuring the mouse is fully anesthetized by toe or tail pinch, place it in a prone position on the platform. Position the lower leg vertically between two loading fixtures. Fit the foot into the cutout of the top fixture and the knee in the cup of the bottom fixture.
Manually adjust the height of the bottom fixture to apply a preload of one to two Newton and tighten the set screw to hold the mouse in position. Next, apply a single compressive load to a target force of 12 to 15 Newton. Set the loading rate in the control file and confirm using the force displacement data.
If a slower loading rate is applied, stop the compressive load immediately after injury to avoid any damage to other joint tissues. Prepare a 29 gauge insulin syringe to inject fluorescence activatable probe solution. Place the anesthetized mouse on its side with the snout in a nose cone.
With the non injecting hand, gently retract the skin around the eye to stabilize the head and protrude the eye. Using the injecting hand, angle the bevel of the syringe towards the eye. Angle the syringe parallel to the mouse's body and proceed by cautiously advancing the syringe beyond the eye until rigid resistance is met.
Slowly inject the probe solution into the retro-orbital sinus and pull the needle from the eye socket. Finally, apply ointment to the injected eye.
