Alik:Paraffin embedding

Materials

 * 80% EtOH
 * 95% EtOH
 * 100% EtOH
 * Hemo-De
 * Paraffin Wax
 * Omni tissue cassettes
 * Forceps

Preparation

 * 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)

 * 1) Place cell/polymer construct in tissue cassette.
 * 2) Place cassette in 80% EtOH for 1 hour.
 * 3) Place in 95% EtOH for 1 hour.  Repeat.
 * 4) Place in 100% EtOH for 1 hour.  Repeat.
 * 5) Place in 50% by volume 100% EtOH / 50% Hemo-De for 1 hour.  Repeat.
 * 6) Place in 100% Hemo-De for 1 hour.  Repeat.
 * 7) Place in paraffin wax at 58°C for one hour.
 * 8) Transfer to more paraffin wax at 58°C for 24 hrs.

Embedding Sample (Day 2)

 * 1) Remove cassettes from paraffin and place in cassette bath of the Leica PEC
 * 2) Using forceps remove cassettes from bath, open, and remove sample
 * 3) Place a metal mold under paraffin dispenser and add a small amount of wax to mold using foot peddle to dispense
 * 4) Position sample as desired in wax, and cover with more paraffin
 * 5) Place mold on cold plate, once fully cooled remove embedded sample from mold. Place samples in freezer until ready to section.
 * 6) Section with microtome at 5-10 µm.
 * 7) 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.
 * 8) Before staining place slide(s) on slide warmer for 45 minutes at 60°C.
 * 9) Stain as desired.