Main Olfactory Epithelium/Bulb Extraction Protocol

Main Olfactory Epithelium/Bulb Extraction Protocol

Original protocol written 6/8/2018 by ECJ

Updated 5/5/26 by CWL

Materials

• Absorbent underpad
• Biohazard bag
• Transfer pipettes
• Surgical tools:
  • Small surgical scissors
  • Serrated forceps
  • Fine angled forceps
• Face shield, or mask and goggles
• Chilled 1X PBS

Setup

1. Prepare extraction surface (on your bench)
   1. Lay out clean tools on absorbent pads (surgical scissors, serrated forceps, fine angled forceps)
   2. Transfer pipettes
   3. Line a waste container (e.g. beaker) with a small biohazard bag

Procedure:

General note: keep tissue moisturized with chilled PBS (can use PBS w/ heparin if you have some leftover from perfusion)

1. Using surgical scissors, cut directly down the midline of the scalp, all the way to the very tip of the nose
2. Peel away flaps of skin on either side of the skull, pulling out the eyeballs. Leave enough of these skin flaps intact so that you have something to hold during extraction
3. Remove the eyes and optic nerves so that you can cleanly see the inside of the orbitals
4. Cut off the back portion of the skull and brain with the surgical scissors, leaving about ½ cm of PFC behind the bulb. You can discard the remains of the skull, cortex, and cerebellum into your waste container
5. Use the surgical scissors to cut to the left and right of the bulb into the eye sockets, then cut straight down the midline of the skull to the bulbs
6. Use the serrated forceps to begin peeling off pieces of skull forward and laterally, from back to front. Periodically drench the tissue with PBS to minimize the tissue sticking to the bone
7. Keep chipping away until the bulbs are fully exposed. Be careful not to crush the bulbs or separate them from the epithelium 
8. Using the fine angled forceps, carefully pull up the back of one of the bones covering zone 1 of the MOE (the long pieces going anterior to posterior over the nostrils). To minimize ripping, try to remove one at a time. If you pull up both at the same time, it’s harder to control
   1. This is the step that usually results in the loss of or damage to your important zone 1 tissue; this will require lots of practice, so be patient and careful
9. Once zone 1 is exposed, continue using the fine angled forceps to remove the super-thin sheets of bone covering the right and left sides of the epithelium (inside the orbital sockets)
10. Then, switch back to the thicker serrated forceps and start chipping away at the bone still covering the top (dorsal) part of the MOE until the entire tissue is fully exposed
11. Use small scissors to cut the very tip (most anterior part) of the MOE and separate it from the tissue at the tip of the snout
12. When you are sure that the tissue is fully exposed, wet the tissue once more with PBS, use the fine forceps to get underneath the bulbs, and grasp the base of the epithelium. Move side to side carefully to loosen the tissue, very gently lift upwards and tilt forwards, and you should feel the epithelium separate 
    1. This is to dislodge the small piece of bone beneath the MOE that is keeping it attached to the rest of the skull. Once you remove this bone, you can cleanly remove the MOE and front teeth from the rest of the skull 
13. To remove the MOE from the front teeth, use the forceps to push the MOE up and out of the teeth
14. The salivary glands may stick to the MOE, or they may detach when you separate the MOE from the teeth. If they stick to the MOE, pull them off with your forceps – they should come off pretty easily
15. Place your extracted sample directly into a 5 ml Eppendorf tube filled with 4% PFA
16. Once all samples have been extracted, cover the tubes (from light) and fix them in PFA overnight or for 1h at 4C (depending on your next protocol)