Hi
I am going to slice paraffin embedded spinal cord. I have two questions:
1. How do I put the SC in the paraffin block so that it will be in coronal orientation? It is not trivial as the part of the SC which has to touch the block, in this case, is very thin and not wide like brain.
2. Can I recognize the ventral horn by Nissl staining (or other staining method)? Can I do this after In situ hybridization?
Thanks
Hi Iddo.
Rat SCs can be placed upright in the mold. This is easy, since completely dehydrated SCs are getting hard and can be handled easier. Nevertheless you should fixate the SC with forceps, until the paraffin is getting hard. For distinction between proximal/distal, rotral/caudal the distal part can be labeled by cutting an edge in the distal/rostral part.
For mouse SCs, the tissue is covered with aluminum foil in order to avoid flection in dehydrating steps. The dehydrated (and unpacked) mouse SC can be placed at the bottom of the mold (lying horizontally, proximal part touching the mold wall) and carefully covered with paraffin (use forceps to hold the SCs in the right position). You can place multiple SC pieces in one block. Before cutting the slices you can remove the paraffin around the SCs carefully and place the block accordingly.
In all cases the vetral horns are recognized easily, because they are thicker (especially in the Th-segment) and motoneurons are the largest cells in the gray matter.
Regarding ISH: I can´t help you in this. Maybe another fellow has some productive suggestions?
Kind regards and good luck.
Hello,
using Nissl-staining you will surely recognize the ventral horn, due to the large alpha-motoneurons. It should be possible to stain Nissl after the in situ, vice versa is not a good approach.
Kind regards
Stefan
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