Table 3.

Troubleshooting LDPI.

Problem	Possible reason	Solution
Poor LDPI signal quality	Insufficient skin area for adequate measurements	• Gently remove hindlimb hair, e.g., by using depilatory creme • Avoid shaving as excessive mechanical manipulation can significantly alter skin perfusion
Changing LDPI signal	Changes in light conditions	• Try to maintain the same light conditions throughout the experiments and adjust the threshold to the background light conditions • Maintain distance of the detector to the hindlimb at the exact same position (typically 20 cm)
Changing LDPI signal	Poor tissue perfusion due to changes in ambient temperature	• Maintain the exact same temperature throughout the experiment particularly avoiding cooling draughts • A closed room for experiments has been proven to be superior for LDPI experimental outcomes
No recovery of perfusion signal	• Cuff too small • Hindlimb has moved	• Change to larger cuffs • Fix hindlimb properly