| wax-sectioning | | 点击： 作者： 来源： 时间： 2007-04-10 本站论坛 |
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- Fix samplesin MEMFA and then into Dent's Fix (20% DMSO/80% Methanol)
- If specimens need to be bleached, bleach in Curis Bleach for 1/2 hour under white light. Store specimens at -20°C in 100% methanol
- place embryos into 100% ethanol (dehydrated with molecular sieve (type 3A - BioRad)
- infiltrate specimens with parafin -- place embryos into glass petri dish containing molten paraplast (58°-59°C). Repeat with fresh molten wax.
- fill a base mold with 5mm parafin -- move embryos into mold with pipette
- 2X washes with xylene, 15 minutes each
- Add 50% EtOH and 50% Hemo-De, 20 minutes for large specimens
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- Replace with 100% wax, 2 hours at 58°C
- Replace with 100% wax, overnight at 58°C *All times are approximate. Large samples require longer time.
- section
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- Heat steel embedding dish over flame briefly before adding wax (this prevents bubbles from forming and allows even cooling of the wax)
- Place enough wax to fill embedding dish
- Add specimen to dish and orient into desired position
- Place embedding ring on the embedding dish (make sure that there is enough wax in the dish so that the embedding ring becomes slightly immersed in the wax - this holds the ring in place)
- Add hot wax to the dish until it nearly reaches the top of the embedding ring
- Carefully reorient the specimen if it has moved
- Cool on cool surface, overnight
- Remove dish and section
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