All of the following procedures are to be performed in a hood or a well vented tissue processor.
1. Fresh nerve tissue received by the Pathology laboratory, usually stretched between two hypodermic needles on dental wax, or in tissue blocks in 4% buffered glutaraldehyde, should be left for at least two hours at 4º C. After consultation with the attending Neuropathologist, cut and processes at least four 1mm. disc-shaped cross-section blocks from the center portion of the nerve. The remaining pieces should be 5mm. long or greater. Depending upon the size of the specimen the Neuropathologist may elect to have either the large or small pieces, or both processed.
2. After two or more hours place the tissue in 0.1M sodium cacodylate buffer at 4º C. for at least 15 min., or store up to a week.
The remainder of this procedure may be performed in a 5 ml. glass container in a refrigerator or on a rotating stand or in a RMC EMP 5160 Ultraprocessor Tissue Processor .
3. Make three changes of 0.1M sodium cacodylate buffer for at least 15 min. each at 4º C.
3.a. Tissue for teasing should proceed to step 4.a. as soon as possible.
4. Place in a covered container in 2% osmium tetroxide with 0.1M sodium cacodylate buffer for two hours at 4º C.
4.a. Place in a covered container in 2% osmium tetroxide with 0.1M sodium cacodylate buffer on a rotating stand in the hood for 24 hrs. Proceed to “Teasing Prep Procedures” below.
4. Place in a covered container in 2% osmium tetroxide with 0.1M sodium cacodylate buffer for two hours at 4º C.
The remainder of this procedure may be done in an uncovered container at room temperature.
5. Place in 0.1M sodium cacodylate buffer for 5 min..
6. Place in in 50% ethanol/distilled water for 5 min.
7. Place in in 70% ethanol/distilled water for 10 min.
8. Place in in 90% ethanol/ distilled water for 10 min.
9. Place in three changes,20 min. each 100% reagent grade ethanol.
10. Place in 50% L.R.White/100% reagent grade ethanol for 30 min.
11. Place in 75% L.R. White/100% reagent grade ethanol for 30 min.
12. Place in two changes,30 min. each in 100% L.R. White
13. Place the large pieces in fresh LR White and refrigerate until delivery to a Neuropathogist for teasing. Embed the disc-shaped pieces in L. R. White in covered gelatin capsules with the nerve tissue disc flat on the capsule bottoms resulting in cross-sections. Allow to stand overnight at room temperature (20º C) in the hood.
14. Place in a 60º oven for 22 to 26 hours.
Teasing Prep Procedures (from 4a above)
5. Place in 20% glycerin in double distilled water for 3 up to 24 hrs.
6. Place in 40% glycerin in double distilled water for 3 up to 24 hrs
7. Place in 60% glycerin in double distilled water for 3 up to 24 hrs.
8. Place in 80% glycerin in double distilled water for 3 up to 24 hrs.
9. Place in 100% glycerin for at least 3 hrs, or deliver to the appropriate person at DHMC Pathology for teasing.
reviewed 8-24-06 CPD
