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. 2015 Jan 31;2015:471216. doi: 10.1155/2015/471216

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