Alik:Paraffin embedding

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Contents


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.

Method

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.

Notes

  • 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 [1])

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