wax-sectioning staining

互联网2013-09-06

686

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
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

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