- 80% EtOH
- 95% EtOH
- 100% EtOH
- Paraffin Wax
- Omni tissue cassettes
- Day 1: Fill a container with paraffin pellets and place in warm water bath, 58 ° C, will take approximately 2-4 hrs for wax to melt
- Day 2: Turn on Leica EG1160 Paraffin Embedding Center (refer to Leica manual located next to machine) and make sure paraffin reservoir is filled with paraffin pellets. The machine needs approximately 4 hrs to warm-up prior to operation.
Sample Dehydration (Day 1)
- Place cell/polymer construct in tissue cassette.
- Place cassette in 80% EtOH for 1 hour.
- Place in 95% EtOH for 1 hour. Repeat.
- Place in 100% EtOH for 1 hour. Repeat.
- Place in 50% by volume 100% EtOH / 50% Hemo-De for 1 hour. Repeat.
- Place in 100% Hemo-De for 1 hour. Repeat.
- Place in paraffin wax at 58°C for one hour.
- Transfer to more paraffin wax at 58°C for 24 hrs.
Embedding Sample (Day 2)
- Remove cassettes from paraffin and place in cassette bath of the Leica PEC
- Using forceps remove cassettes from bath, open, and remove sample
- Place a metal mold under paraffin dispenser and add a small amount of wax to mold using foot peddle to dispense
- Position sample as desired in wax, and cover with more paraffin
- Place mold on cold plate, once fully cooled remove embedded sample from mold. Place samples in freezer until ready to section.
- Section with microtome at 5-10 µm.
- Place “tapes” of sections on colorfrost slide with 30% EtOH, and place slide on slide warmer (60°C) for 1 min. Remove and leave out to dry at room temperature overnight.
- Before staining place slide(s) on slide warmer for 45 minutes at 60°C.
- Stain as desired.
- Hemo-De is also known as Citrisolv, and is purchased from Fisher (22-143-975)
- Paraffin wax is Paramat Extra from Electron Microscopy Sciences
- Use pencil to mark cassettes as marker will wash off in Citrisolv
- Dehydration steps can be broken up into two days by leaving the cassettes in the second 100% EtOH bath overnight
(based on the PDF )