Table 1 |.

Troubleshooting table

Step	Problem	Possible reason	Solution
1	Mouse is not fully anesthetized	•Anesthetic dose was insufficient	Increase the dose of anesthesia
		• Different strains of mice may have different sensitivities to anesthesia
	Mouse dies while under anesthesia	• Different strains of mice may have different sensitivities to anesthesia	Anesthesia should be carefully titrated and monitored with respiratory gating for the duration of tumor implantation, imaging, and neurological function tests
		• When mice become moribund at the end stage, they may be more sensitive to anesthesia
	Mouse experiences hypothermia	The mouse cannot maintain stable body	Use a rectal temperature probe to monitor the body temperature and initiate appropriate actions that stabilize core body temperature, such as applying a heating pad
		temperature during general anesthesia
5,8,9	Bleeding from the skull	Injury to blood vessel	Provide hemostasis with Gelfoam, and then rinse with sterile saline
11	Difficulty in detaching tumor cells from cell culture flask	Trypsin condition is not optimal	Rinse the flask with PBS to completely remove the FBS
			• Increase the amount of trypsin–EDTA
			• Incubate the trypsin reaction in a 37 °C cell culture incubator
20	Tumor cell leak during the injection	Injection and retraction of the needle were performed too quickly	Inject the cells slowly (over 30 s), hold the syringe for 60 s, and retract over 30 s
30	Low GLuc reading	• GLuc reporter gene level is low in tumor cells	• Repeat in vitro cell culture and counting
			• Test the cells for mycoplasma contamination
		• GLuc assay is not optimal	• Re-infect tumor cells with GLuc virus
			• Repeat sorting/selection of GLuc-transduced tumor cells
			• Optimize GLuc assay, including use of a new kit and checking the luminometer setting
31	Hearing loss in mice bearing cranial window	The surgical procedure to implant the cranial window can cause an accumulation of secretions in the external auditory canal	Use mice without cranial windows for hearing testing
37	High rejection rate during the hearing test (strong background activity)	Electrodes are positioned incorrectly or anesthetic depth is inadequate	Reposition the electrodes or provide anesthetic boost as needed
42	Large variation between DPOAE and ABR measurements	The positioning of the electrodes is inconsistent and the recording system is not correctly calibrated	Consistently position the electrodes at the same anatomical location and calibrate the system before testing
45A(v)	Cerebellar window becomes loose or dislodged	Too much force may have been used when fixing a window for imaging	Handle the mouse gently when fixing the window and ensure that the mouse is anesthetized while imaging
	Tumor burden is not detectable by IVM	Tumors may be too small or tumors are implanted too deeply to image	Carefully scan the areas implanted with tumor cells or wait until tumors reach a larger size to image
45B(i)	Blurry image	• Tissue is outside the focal plane	• Adjust the distance between the tissue and the lens
		• Coverslip is dirty
			• Clean the coverslip with an alcohol swab
45B(ii)	Vessels appear in one side of the tissue but not the other	Tissue is not level	• Adjust the animal to level the tissue
			• Use a surface normalization algorithm
45B(iv)	Vessel appearance and clarity differ between the tumor and the surrounding tissue	Tumor growth leads to changes in tissue topography	Image the tissue twice, both at the tumor plane and in the tissue plane