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 |
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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. |
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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) |
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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 |
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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 |
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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 |
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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 |