Recommended materials and stimuli

Item	Specifications	Purpose	Source
Mesh-top platform	The mesh should contain small holes around a half centimeter in width to allow for stimulus access. We used super-corrosion resistant stainless steel purchased from McMaster-Carr (product no: 1337T93). The platform should be high enough (~12–16 inches) to allow for the experimenter to have access from below	Mice will be placed on this platform to gain access to their paws from underneath	Machine Shop (custom made)
5-sided rectangular plexiglass mouse chamber	Container should measure ~4.25 cm tall × 4.5 cm deep × 11.5 cm long. The thickness of the plexiglass used was 0.25 cm. It is essential for this restrainer to be rectangular because it positions the rodent’s paw perpendicular to the camera when recording as opposed to circular restrainers which allow the rodent to face any direction	Individual mice will be placed in this restrainer during experimentation	Machine Shop (custom made)
High-speed camera w/ tripod	There is a range of high-speed cameras on the market. We recommend a camera that can record 2–3 s of 500–2,000 fps. We have successfully resolved all behaviors at 500 fps. To determine whether a particular camera is suitable for this method, we recommend recording a video and simply confirming whether all behavioral features can be resolved both temporally and spatially	Camera for recording high-speed videos of mouse reactions	We use the FastCAM UX100 800K-M-4GB - Monochrome 800K with 4 GB memory for our recordings, but other cameras, even low-cost cameras, are available and should be sufficient. We recommend testing any camera to confirm all sub-second behavioral features can be resolves
Red or infrared light	If using infrared light, be sure the camera used is IR-sensitive	To decrease the disturbance of rodents, we recommend lighting the behavior room with red or infrared light	CMVision IP65
Recommended stimuli
Cotton swab	To simulate an innocuous “static touch” stimulus, we use a cone-shaped cotton swab (do not use a pointed cotton swab)	This stimulus should be applied to the plantar surface of the hind paw through the mesh briefly (<1 s). Ensure sufficient pressure is placed to allow the cotton swab to move past the mesh and make contact with the rodent’s paw, only part of the Q-tip will pass the mesh and touch the paw	Q-Tip Cotton Swabs that can be purchased from any pharmacy store
Makeup brush	To simulate an innocuous “dynamic touch” stimulus, we use a makeup brush	Gently brush the plantar surface of the hind paw in a proximal to distal (i.e., heel to toe) direction for the length of the hind paw with the brush. The brush should be moved at a moderate speed (<1 s) and should be consistent between animals	e.l.f. Foundation Makeup Brush purchased at CVS
Metal needle	To simulate a noxious stimulus, we apply a small metal needle to the mouse’s foot	Apply the needle to the plantar surface of the hind paw at two different speeds/forces (light & heavy pinprick). Both pinprick forces should not push the rodent’s paw upward and should instead stop at or close to the mesh surface. Pushing the needle through too far can injure the animal or obscure the height measurement during analysis. This can be confirmed in the video. To simulate the “light pinprick,” gently raise the needle until it comes into contact with and puts pressure on the paw and then remove the stimulus. To simulate “heavy pinprick,” quickly and forcefully poke the paw	Austerlitz Insect Pins (size 6, 38 mm length, 0.65 mm diameter)
von Frey Hairs	To simulate a range of mechanical forces, we have used VFHs	From our analysis, we have found that 4 g induces a movement similar to that which is seen with heavy pinprick. 1.4 g induces a movement similar to that which is seen with light pinprick and 0.6 induces a movement similar to that which is seen with dynamic brush (i.e., 4 g likely induces pain, 1.4 g border-line pain, and 0.6 g non-painful)	Von Frey Hairs, Stoelting Company, 58011