Paraffin embedding tissue

Purpose

To embed tissue in paraffin

Protocol

  • Fix tissue in fixative of choice (normally 4% paraformaldehyde or 10% buffered formalin) overnight 4 °C.
  • Wash 1 or 2 times in PBS.
  • Fix tissue in 70% ethanol overnight room temp. Can leave at this stage forever.
  • Dehydrate tissue either manually or using the tissue processor in Largaespada lab.

Manual :

  • 45 minutes 70% ethanol
  • 45 minutes 80% ethanol
  • 45 minutes 95% ethanol
  • 45 minutes 100% ethanol
  • 60 minutes 100% ethanol
  • 60 minutes 100% ethanol
  • 60 minutes clearing agent (xylene or citrosol)
  • 60 minutes clearing agent (xylene or citrosol)
  • Place tissue in tissue cassettes and place in vacuum oven at 60°C overnight.
  • Remove from oven and let cool slowly
  • Remove tissue cassette and mount on cutting block and proceed to sectioning

Using the Tissue Processor in Largaespada lab:

  • Fill or top-off all 12 reagent cans (~1.7 l). Reagent level should be between two indented lines.
  • Change PBS if it appears moldy. (bring 10x PBS to the machine to fill).
  • Readout should say U01 with a time.
  • Press Start, then use the + or – key to select the program you want.
  • Press start again.
  • When program finishes, press up key and wipe paraffin off metal basket right away with a paper towel.
  • Up and down arrows moves revolving head up and down.
  • Lock key will lock operation, just hold down for 5 sec to lock or unlock.
  • Clock key will tell you how much time is remaining in the program.
  • Three arrow key is to vibrate, this should always be on.

Notes:

Use Omnisette tissue cassettes for easiest operation.

Also use the Metal slide holder in combination with the Tissue Path embedding ring (Cat# 22038201) to mount.