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All of the following procedures are to be performed in a hood or a
well vented tissue processor.
1. Fresh muscle tissue received by the Pathology laboratory on a stretching
forceps or in tissue blocks in 4% glutaraldehyde buffered with either 0.1
M Pipes buffer or 0.1M sodium cacodylate buffer should be left
for at least two hours at 4º C., cut into muscle fiber cross-section blocks and
placed into a covered container containing 4% buffered glutaraldehyde at 4º
C.
2. After two or more hours place the tissue in 0.1M sodium cacodylate
buffer at 4º C. for at least 15 min. 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.
4. Place in a covered container of 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. Embed in L. R. White in covered gelatin capsules, with the fibers in
cross-section. Leave overnight at room temperature in the
fume hood to ensure full infiltration.
reviewed 8-24-06 CPD
14. Place in a 60º oven for 22 to 26 hours.
reviewed 8-24-06 CPD
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