Protocols:
Olfactory epithelium explant protocol.
Day 1: Prepare the substrates.
Coat 8 well chambered coverglasses with 50 l of 50 g/ml poly-D-lysine in
sterile dH2O.
Incubate ON at RT in the fume hood.
Day 2: Finish substrate preparation:
Wash 3 x 5 min in sterile dH2O (trying not to scratch the PDL layer).
Air dry open in the fume hood. (can be stored for a while before use).
Cover with the molecule of interest. Add 50 l 20 g/ml Laminin diluted in sterile
dH2O, incubate for 60 min at 37 C in 5% CO2 (cover in the incubator)
Wash 3 x 5 min in sterile dH2O (and leave it with the water until it is ready to
plate de explants). Don’t let it dry!
Preparing the Media (preferentially the week or the day before):
500 ml Neurobasal (Gibco)
10 ml 50X B-27 supplement (Gibco)
5 ml 100X Pen Strep
5 ml 200 mM L-Glutamine
NB Supplemented media is good for approximately 4 weeks at 4 C, and should
then be discarded.
Earle’s Balanced Salt Solution
Preparing the Albumin ovomucoid inhibitor:
First thing: add 32 ml of Earle’s Buffered Salt Solution (EBSS) to the albumin
ovomucoid inhibitor mixture and allow the components to dissolve, by gently stirrer. And
allow it to equilibrate (check the pH color with the indicator) open in the fume hood for
an hour (or so).
Harvesting and culturing explants:
Rapidly decapitate 6 P1 pups. Cut (in order) the jaw, the front part of the nose,
and the back of the brain. Peel the rest of the head, and leave it in a dish with PBS 1X
(Gibco).
Dissect and collect the OEs. Before using any dissecting tool put into de sterilized
beads for a few seconds. Briefly grab the head by the eyes with N2 forceps, and with a
pair of fine dissection scissors cut the skull between the hemispheres up to the OBs. Cut
the skull between the OB and the hemispheres. With N5 forceps take out the skull and
then remove the OBs and the brain. Cut all the nerves beneath the OBs. Cut at both sides
of the nasal septum with the scissors, and then with a scalpel take out the rest of the head.
Keep the nasal septum, and cut in the borderline between respiratory and olfactory
epithelium (it can be seen some small lines). With a forceps (closed) take out the OE
from both sides of the septum, and put it into a 15 ml Falcon tube with 5 ml of Hank’s
Balanced Salt Solution (HBSS).
Aspirate the supernatant (take care not to aspirate the explants)
Resuspend it in activated papain (Worthington Papain Dissociation Kit). For this
add 5 ml of EBSS to papain vial and place it in a bath at 37ºC (or at RT). Add 250 l of
DNase solution (in the kit).
Displace air with 95% O2 / 5% CO2 and cap immediately.
Incubate on nutator for 5 min at 37 C + 5% CO2.
Triturate gently using a siliconized fire-polished Pasteur pipette (16 times).
Spin 5 min at 1.5K
Aspirate the supernatant.
Resuspend in a solution containing 2.7 ml EBSS + 300 l of reconstituted
albumin inhibitor solution + 150 l DNAse solution (add 500 l of EBSS to the DNase
vial) (Worthington Papain Dissociation Kit).
Overlay on 5 ml Albumin inhibitor. (It’s a gradient)
Spin 5 min. at 1.5K. Dissociated cells pellet at the bottom of the tube, membrane
fragments remain at the interface.
Aspirate the supernatant.
Resuspend in 10 ml supplemented media and transfer to 15 ml TC dish.
Select explants in 50 l media (with an automatic pipette) and plate it on a well.
Incubate ON at 37 ºC + 5% CO2 (on the incubator).
Optative: flood cells with media. It can be done next day.
Day 3:
If it wasn’t done the previous day, flood cells with media.
Return to the incubator and leave it at 37 ºC + 5% CO2.
Fixing the explants:
After 48 hrs (or the time needed), aspirate the media, but don’t remove the media
on the wells, rinse cells with dPBS (to remove the media).
Fix in 4% PFA and 4% sucrose in PBS for 30 min at RT.
Wash 3 x PBS.
Store in PBS at 4 ºC before staining.
Day4: Staining
Take out the silicone from the 8 well chambered coverglasses, and placed them on
a slide microscope and sitck it with a silicone square. Don’t let the explants dry. Add a
silicone rectangle in order to limit the liquid you add to the wells. And fill it with the PBS
they were submerged in.
Remove the PBS with a tip attached to a vacuum, without touching the explants,
and add 500 µl of 2% BSA in TBS-T, for 15 min.
Remove the BSA and add 500 µl of primary antibody for 60 min on 2% BSA.
Wash 3 x TBS-T.
Add 500 µl of secondary antibody + dyes for 30 min. on 2% BSA.
Wash 2 x TBS-T.
Wash TBS.
Take out the silicones, add Gel Mount on the coverglass and mount on the same
microscope slide.