Dec 13, 2022
Version 2
Iodine-starch sweating assay V.2
- Fanglin Lu1
- 1MD, MS
Protocol Citation: Fanglin Lu 2022. Iodine-starch sweating assay. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l69d1klqe/v2Version created by Fanglin Lu
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 12, 2022
Last Modified: December 13, 2022
Protocol Integer ID: 73852
Keywords: starch sweating, sudomotor function in mice, systemic pilocarpine stimulation, sweating assay, starch reaction, kinds of sweating assay, iodine, restraint stress, sudomotor function
Abstract
This protocol outlines the procedure for assessing the sudomotor function in mice under restraint stress and under systemic pilocarpine stimulation. Both two kinds of sweating assays were based on the iodine-starch reaction.
Guidelines
Please follow the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guideline.
It is favorable to not perform these two different kinds of sweating assays on the same day.
Materials
10% povidone–iodine solution (Yoshida Pharmaceutical Co., Ltd.)
Starch (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan; catalog number: 193-09925)
Caster oil (Sigma-Aldrich, St. Louis, MO; catalog number: 259853)
Mini makeup brushes (we use them instead of cotton buds)
Microscope (Olympus SZ61) with digital camera Moticam 1080
Self-made restrainer (The nose piece is BROKEN. USE A NEW ONE.)
Note
The restrainer is made of a nose piece of commercially available mouse handling tube and a 50mL syringe.
Mouse will bite the nosepiece under restraint stress, and get injured by the broken nosepiece.
Change the nose piece once it is not intact. Or use round nylon nose piece, which is safer.
Pilocarpine hydrochloride (Sigma-Aldrich; catalog number: PHR1493)
Troubleshooting
Before start
Check if the restraint tube is intact.
Prepare 100% starch–castor oil (1 g starch per 1 mL castor oil)
Prepare pilocarpine (diluted in 150 µL 0.9% saline).
Restraint stress-induced sweating
6m
The mouse is immobilized in a self-made restraint tube.
self-made restraint tube
2m
Apply 10% povidone–iodine solution to the plantar surface of bilateral hind paws with a cotton bud. Once dry, coat the skin surface with a suspension of 100% starch–castor oil.
1m
Changes on footpads are video-recorded for 3 min. The number of dark spots was counted for each paw.
Expected result
Sweat droplets are visualized as dark-colored spots because of iodine–starch reactions.
3m
Pilocarpine-induced sweating
7m 10s
The mouse is placed in the supine position under sevoflurane anesthesia (4 volume% in 1 L/min oxygen).
Note
Sevoflurane inhalation was used, as it is safe, non-invasive, rapid, and easy to control.
Adequate anesthetic depth was ascertained by loss of response to tail clamp.
1m
Apply 10% povidone–iodine solution to the plantar surface of bilateral hind paws with a cotton bud. Once dry, coat the skin surface with a suspension of 100% starch–castor oil.
1m
Inject 100 µg pilocarpine hydrochloride intraperitoneally.150 µL diluted in 0.9% saline
10s
Changes on footpads are video-recorded for 5 min. The number of dark spots was counted for each paw.
Expected result
5m