A Fluorescent Dye Method Suitable for Visualization of One or More Rat Whiskers

[Abstract] Visualization and tracking of the facial whiskers is critical to many studies of rodent behavior. High-speed videography is the most robust methodology for characterizing whisker kinematics, but whisker visualization is challenging due to the low contrast of the whisker against its background. Recently, we showed that fluorescent dye(s) can be applied to enhance visualization and tracking of whisker(s) (Rigosa et al., 2017), and this protocol provides additional details on the technique.

Before starting this protocol, the rat should be sedated (see Procedure B: 'Animal sedation': follow the 'pre-anesthesia' and 'anesthesia' procedures).
1. Place the rat on the heating pad and set the body temperature to 37 °C.
2. Put the Epigel on the rat's eyes to protect them from light and dryness.
3. Apply the discoloration preparation ( Figure 1A) on all whiskers using cotton swabs. Since its smell is quite strong, avoid placing it close to the rat's nose, because breathing it can reverse 4 www.bio-protocol.org/e2749

Wait between 30 min and 1 h to bleach whiskers.
5. Rinse the whiskers using distilled water with a cotton pad ( Figure 1B). 6. Dry the whiskers with air-flow or a cotton pad ( Figure 1C). 7. Place the rat's head under illumination and tilt it a bit to reach the whisker(s) you are interested in.
8. Isolate selected whiskers ( Figure 1D) a. Cut a piece of Parafilm to cover the rat's snout and cheeks (e.g., 6 cm L x 6 cm H).
b. Make a small hole to let the whisker pass through. You can use a glass capillary or a tweezer to isolate the whisker, in both cases paying attention not to damage the whisker or the rat's skin.
c. The resulting working surface allows application of the dye(s) only on a selected subset of whiskers. This is very important in order to prevent the unwanted application of the dye. 9. Flatten the working surface at the base of the selected whisker(s) to allow the application of the dye on the complete whisker(s) length.
10. Apply the fluorescent dye on whisker(s) ( Figure 1E). For each whisker: a. Cut a piece of Parafilm of circa 2 cm width and slightly longer than the whisker. b. Fill the syringe with the fluorescent dye and eject it abundantly onto the Parafilm in order to soak the whisker along its entire length. In order to avoid air bubbles, which would locally prevent staining, spread the dye with a cotton swab.
c. The whisker now adheres weakly to the Parafilm because soaked in a gel. Any movement can make the whisker slip away. It will be helpful to hold the Paraffin film or even tighten it with aluminum foil. 11. Wait between 30 min and 1 h from the last application to let the dye diffuse inside the cuticle.
12. Remove the excessive dye and rinse the whisker(s) using a cotton pad or a gauze wet with vinegar, which tends to close the cuticle in complete safety for the animal.  c. Visualize the whisker(s) and adjust the camera gain (Figures 2 and 3).
Note: If the background is much darker than the visualized whisker, the gain of the camera sensor can be safely increased without introducing salt and pepper noise to the image. This feature depends on the specific camera model.
14. Follow the guidelines for Post-anesthesia (see Procedure B: 'Animal sedation') to recover the rat.
15. In case the visualization is not sufficient (e.g., the whisker did not absorb the dye along its entire length), plan another application of the dye. In case the time left before the current sedation ends is not sufficient for immediate re-staining, ask your veterinarian when another sedation for the same animal could be feasible.