Table 1.

Troubleshooting: the most frequent problems in preparing and maintaining the organotypic longitudinal spinal cord slice culture, their probable reasons, and their proposed solutions.

Problems	Possible reason	Solutions
Spinal cord destroyed during chopping	The tissue is too soft	All the procedures must be done on ice and the chopper table should be chilled before cutting
	The blade cuts too fast and tears the tissue up	The blade speed should be approximately adjusted to make a single cut every single second
	The blade does not stick closely to the chopper table	Make sure that the blade tightly sticks to the chopper table
Aberrant axonal projections	The spinal cord is not placed in a parallel-to-the-blade fashion	Before cutting check whether the spinal cord is set parallel to the blade.
Slices die after 1 week	Incorrect pH of the medium	pH must be adjusted to 7.2 each time the medium is changed
	The precutting procedure takes too much time	The whole procedure should not take more than 90 min (from decapitating animals up to placing the slices onto the membrane)
Transplanted cells die after short time	The density of the transplanted cells is too high	Try different amount of cells
	The cells are not evenly suspended	Before transplantation mix cells in eppendorf with pipette gently but precisely
Slices and cells detach from the membrane during fixation	PFA was old or too cold	After preparing PFA solution do not freeze the preused doses and before fixation heat PFA up to 37°C
Slices are not evenly stained	The permeabilization is too weak or too short	Add Triton-X 0.2% to the primary antibody solution
	The slice was not completely covered with liquid	Using a brush or tips let the slice gently sink to the bottom of the well; it should stay there during entire staining procedure
The slice structure is destroyed during slide closing	The coverslip is too heavy and crushes are rich in lipids structure	Add a double portion of mounting medium and wait a while until it dries a little before applying coverslips