Materials and Reagents

Cotton
75% Ethanol
Ketamine hydrochloride (100 mg/ml) (Ketaject, Phoenix Pharmaceuticals, NDC number: 57319-542-02 )
Xylazine hydrochloride (100 mg/ml) (AnaSed Injection, LLOYD, NADA number: 139-236 )
Xylocaine (lidocaine hydrochloride) 2% Jelly (AstraZeneca, NDC number: 0186-0330-01 )
Bacitracin zinc ointment (USP 500 U/g) (Fougera Pharmaceuticals, NDC number: 0168-0011-31 )
Saline (0.9% sodium chloride injection USP)

Equipment

Hood
Autoclave
Aluminum foil
Dumont #5 forcep (Fine Science Tools, catalog number: 11254-20 )
Dumont #3c forcep (Fine Science Tools, catalog number: 11231-20 )
Cohan-Vannas spring scissor (Fine Science Tools, catalog number: 15000-02 )
Fine scissor (Fine Science Tools, catalog number: 14060-09 )
UNIFY Silk surgical suture [small (P-3) 13 mm, reverse cutting 3/8 circle needle. 4-0 18"/45 cm thread] (AD Surgical)
Alcohol pad (PDI, catalog number: B603 )
Gloves
Microscope
Illuminator (AmScope, catalog number: Model HL250-AY )
Heating pad (Harvard Apparatus, catalog number: 507220F )

Procedure

One day before the surgery
1. Sterilize the hood with ultraviolet germicidal irradiation for 1 h.
2. Autoclave the surgical tools wrapped with aluminum foil, some cotton wrapped with aluminum foil and another N+1 (N is the number of mice to be sutured) aluminum foil. Surgical tools include two Dumont #5 forceps, one Dumont #3c forcep, one Cohan-Vannas spring scissor and one fine scissor.
3. Sterilize the suture needle and thread with 75% ethanol one day before the surgery.
On the day of surgery
1. Put all the surgical tools along with some alcohol pads in the sterilized hood.
2. Anesthetize the mouse with the mixture of 100 mg/kg ketamine hydrochloride and 10 mg/kg xylazine hydrochloride intraperitoneally.
3. Clean the mouse with alcohol pads, and then put it on one sterilized aluminum foil. Put all the tools on another foil. Spray the gloves with 75% ethanol.
4. Under microscope and illuminator, suture the eyelids using sterilized needle and thread with the help of Dumont #5 forceps as follow: make 2-3 stitches, tighten the thread, make two knots, and cut the thread with the fine scissor as short as possible. For monocular deprivation of more than three days, cut the eyelashes and the edge of eyelids using the spring scissors before suturing. In case of bleeding, stop it with the cotton before continuing.
5. Apply a thin layer of xylocaine 2% Jelly and bacitracin zinc ointment to the sutured eyelids.
6. Put the mouse on the heating pad.
7. Spray the surgical tools with 75% ethanol, clean it with alcohol pads, and put it back on the aluminum foil. Dispose the dirty aluminum foil used for the mouse.
8. Repeat procedure 2-7 for other mice.
9. Put all the mice back into the holding cages after making sure they are fully recovered.
10. Clean all the surgical tools and the hood with 75% ethanol. Sterilize the hood with ultraviolet germicidal irradiation for at least an hour.
After monocular deprivation
1. At the end of the deprivation period, mice were anesthetized, stitches were removed, and lid margins were separated. Eyes were flushed with sterile saline and checked for clarity under a microscope. Only mice without corneal opacities or signs of infection were used.

Acknowledgments

This protocol is adapted from a published paper: Ma et al. (2013). This work was supported by grants to H.W.T. from the National Institutes of Health (EY019049 and EY022478) and the Kirchgessner Foundation.

References

Hubel, D. H. and Wiesel, T. N. (1963). Receptive fields of cells in striate cortex of very young, visually inexperienced kittens. J Neurophysiol 26(6): 994-1002.
Ma, W. P., Li, Y. T. and Tao, H. W. (2013). Downregulation of cortical inhibition mediates ocular dominance plasticity during the critical period. J Neurosci 33(27): 11276-11280.