To embed tissue in paraffin
- 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.