Procedure steps no.	Problem	Possible explanation	Solution
2	Mouse with experimental autoimmune encephalomyelitis (EAE) dies after injection of fentanyl/midazolam/medetomidine	The mouse is too ill or lost too much weight during EAE and cannot tolerate anesthesia.	Do not use EAE animals with a clinical score more severe than paraplegia or with weight loss of more than 20%.
2	Animal is not anesthetized after 10–15 min of fentanyl/midazolam/medetomidine injection	Anesthetic mixture was not properly injected intramuscularly.Anesthetic has lost its potency, as it is either expired or has not been freshly prepared.	Repeat administration with a half of the original dosage.Open a new stock or freshly prepare the anesthetic mixture.Store anesthetics in lightproof containers.
7	Observing signs like twitching of whiskers showing that the animal begins to wake up after connection to the ventilator	Diameter of the tracheal cannula is too small for the diameter of the trachea or has not been appropriately fixed with the sutures. Consequently, isoflurane is leaking out.	Make sure that the outside diameter of the tracheal cannula fits tightly within the animal’s trachea and fix appropriately.
8	Overdose of anesthetics	Time interval between the first anesthetic (fentanyl/midazolam/medetomidine) and the second one (isoflurane) is too short.	After connection of the animal to the ventilator, first apply only mixed gas of O2 and N2. Apply isoflurane only after a proper time interval.
10	Irregular electrocardiogram	Animal did not receive a proper volume of anesthetics.The electrodes have been dislocated.	Increase the isoflurane percentage (suggested appropriate level: 0.5–1%).Check the electrodes and be sure that they are connected at the corresponding positions.
9	Body temperature is not stable and temperature controller gives an alarm	Rectal probe for temperature has not been inserted properly.	Check the rectal probe and secure it with tape. Temperature should be set on 35 ± 1°C.
12	Abdomen of the mouse is inflated after turning to the prone position	Trachea or tracheal catheter has been twisted by turning the mouse and blocked the flow of anesthesia gas to the lung.	Repeat the surgery with a new animal. To avoid inflation, turn the animal and all the tubing connections at the same time and in the same direction.
13	Ear bars do not engage the ear canal	Position of animal’s head is not straight.Head is in a higher or lower level compared to the ear bars.	Place the head in a horizontal position in line with the body and avoid bending.Add or remove gauze pads under the belly of the mouse.
15	Bleeding	Damage to the blood vessels during surgery.	Use forceps with an angle and use them gently.
15	Cervical bones are broken while stabilizing the spinal cord	Excessive pressure was applied.	Avoid putting too much pressure on the preparation while stabilizing the bones with rib holders.
16	No observation of cells or blood flow in the microcirculation of the spinal cord window after infusion of cells via the carotid catheter	Cells might have aggregated and blocked blood flow in a blood vessel outside of the regions of interest (ROI).	Check additional fields of view. Repeat with a second injection.If not successful start with a new animal. To avoid aggregation of cells, carefully resuspend cells in 0.9% isotonic NaCl right before infusion to the catheter.
17	Blood flow in dorsal vein is slowed down	Body temperature of the animal is dropping.	Cover the animal with some gauze pads. Check if the rectal temperature probe is properly inserted and whether the temperature controller is adjusted correctly.
21, 23	ROI is not visible under the two-photon microscope	Viscotears® liquid gel is leaking out of the window preparation.	Apply agarose around the whole spinal cord window to avoid leaking of Viscotears®.
18, 19	Fluorescence signals are low	Laser power and the voltage of PMTs are low.Excitation wavelength is not appropriate.Bleeding from surrounding tissue covered the ROI.Agarose has covered ROI.	Increase the laser power and the respective PMT voltages.Change to another excitation wavelength.Clean the blood from ROI with a gauze pad and NaCl.Clean ROI with gauze pad and NaCl.
20	Fluorescence signal of blood vessels is low	Fluorescent dextran dye or fluorescently labeled antibody has leaked from the vessels.	Repeat injection of plasma marker. If there is no improvement repeat the whole surgical procedure with a new animal.