Evicerate embryos, remove head and tail in cold PBS
Fix in 4% PFA on cold room nutator 1 hour
Wash 2-3 x 10 min. with cold PBS on cold room nutator
Leave in PBS on cold room nutator O/N
Replace PBS with 30% sucrose
Nutate in cold room 2 hours
Mount in O.C.T. (remove excess sucrose)
Store at -80°C
Cut sections and collect on labeled slides
Place slides on horizontal trays, dry ~30 min
Add 1 ml PBS to each slide to rinse off OCT
Pour off PBS
Add 0.5 ml per slide 1° antibody solution
Leave O/N in cold room
Wash slides 3 x 5 min. with PBS
Add 2° antibody solution
Cover, room temp for 2 hours
Wash 3 x 5 min. with PBS
Mount coverslip with Vectastain Mounting Media
Store at 4°C
| 50 ml 4% PFA | 1° Ab Soln | 2° Ab Soln. |
|---|---|---|
| 25ml H2O | ||
| 2 drops 10N NaOH | PBS 1 ml | PBS 1 ml |
| HINGS 10 µl | HINGS 10 µl | |
| -heat- | 5% Triton X 20 µl | Triton X 20 µl |
| Ab Rab. T4 0.67 µl (1:1500) | Donkey α Rab.FITC 2 µl (1:500) | |
| Add 2 g PFA | Ab GP HB9 0.25 µl (1:4000) | Donkey αGP cy3 1.3 µl (1:750) |
| -dissolve, cool- | 1 ml | 1 ml |
| Add 25 ml 0.2M PB | ||
| -sterile filter- | ||
| Keep on ice |