Mouse Protocol for Nissl Staining
Experiment re: cochlea removal
Mice receive cochlear ablation (right side) and are allowed to survive for various time points.
Mice are anesthetized with 70 mg/kg of sodium pentobarbitol for perfusion.
Mice are given intracardiac perfusion of 4% paraformaldehyde in PBS. The head is removed, the skull is carefully opened to expose the brain, and the whole head is submerged in 4% paraformaldehyde in PBS for at least 4 days at 4° C.
The brain is carefully removed from the head, blocked (as to remove the anterior portion of the cortex) and notched on the right ventral surface. Rinse in PBS for ~8 hrs. (change PBS several times) or under running tap water for 4-8 hrs., and then placed in 70% EtOH 2 x 1 hr. or overnight at 4° C. Where to block:
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Brainstems are dehydrated: (with slow agitation- on a shaker) |
P15 or younger |
P16 or older |
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95% EtOH |
45 m |
45 m |
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95% EtOH |
45 m |
45 m |
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100% EtOH |
30 m |
45 m |
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100% EtOH |
30 m |
45 m |
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Meth. Salic. |
1 h |
1 h |
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Meth. Salic. |
1 h |
until it sinks |
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Infiltrated with paraffin: (apply ~5 PSI vacuum) |
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Paraplast 1 |
45 m |
45 m |
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Paraplast 2 |
45 m |
45 m |
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Paraplast 3 |
30 m |
45 m |
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And embedded in fresh paraffin in a peel away mold. Allow to cool before sectioning.
Tip: prior to placing brainstem in the mold, fill it ~ half way with paraffin and place in oven to keep warm. When you remove the brainstem from the paraffin pot, place it directly in the warm mold with paraffin, center it and allow it to anchor to the bottom, then fill the mold the rest of the way to the top with paraffin. (Slightly overfill the paraffin, as it retracts while cooling.)