TITLE: Isolation and Processing of Embryonic and Postnatal Brains
AUTHORS: Maryana Nissan, divya.darwinarulseeli

[DESCRIPTION]
This protocol describes:
-Harvesting embryos from females from embryonic day 10 [e10] to embryonic day 18 [e18] 
-Brain isolation of embryonic (e10-e18) and postnatal brains (p0-p30) 
-Processing and Freezing of embryonic and postnatal brains 

[STEPS]
SECTION: Harvest embryos from female mouse from embryonic day 10 (e10) to embryonic day 18 (e18)
1. Place pregnant female in an anesthetic chamber for 2 minutes 
SECTION: Harvest embryos from female mouse from embryonic day 10 (e10) to embryonic day 18 (e18)
2. Euthanize female using cervical dislocation
SECTION: Harvest embryos from female mouse from embryonic day 10 (e10) to embryonic day 18 (e18)
3. Grip the abdominal wall with forceps and make incisions along the region to expose the abdomen 
SECTION: Harvest embryos from female mouse from embryonic day 10 (e10) to embryonic day 18 (e18)
4. Cut away the uterus with scissors
SECTION: Harvest embryos from female mouse from embryonic day 10 (e10) to embryonic day 18 (e18)
5. Place the uterus in a petri dish filled with 1x phosphate-buffered solution [1x PBS] 
SECTION: Harvest embryos from female mouse from embryonic day 10 (e10) to embryonic day 18 (e18)
6. Carefully poke the amniotic sac right next to the placenta to expose the embryo. 
SECTION: Harvest embryos from female mouse from embryonic day 10 (e10) to embryonic day 18 (e18)
7. The embryo will slide out of the sac, do not handle the head of the embryo with the forceps 
SECTION: Harvest embryos from female mouse from embryonic day 10 (e10) to embryonic day 18 (e18)
8. Behead at the neck of the embryo and collect a tail clip into its corresponding falcon. Each falcon should be filled with 4% paraformaldehyde [4% PFA] 
SECTION: Harvest embryos from female mouse from embryonic day 10 (e10) to embryonic day 18 (e18)
9. Repeat to the remaining embryos in the uterus 
SECTION: Harvest embryos from female mouse from embryonic day 10 (e10) to embryonic day 18 (e18)
10. Embryonic heads are left in 4% PFA overnight for fixation 
SECTION: Brain isolation of embryonic and postnatal brains
11. Leave embryonic heads in 4% PFA for one night, and postnatal heads in 4% PFA for two nights 
SECTION: Brain isolation of embryonic and postnatal brains
12. When fixation is complete, transfer the brain from 4% PFA into 1X-PBS 
SECTION: Brain isolation of embryonic and postnatal brains
13. Place petri dish under a dissecting microscope [from Leica Microsystems]
SECTION: Brain isolation of embryonic and postnatal brains
14. Place head in the petri dish 
SECTION: Brain isolation of embryonic and postnatal brains
15. The head is held down and stabilized by inserting the forceps into the eyes
SECTION: Brain isolation of embryonic and postnatal brains
16. Push the forceps inwards, making a medial cut 
SECTION: Brain isolation of embryonic and postnatal brains
17. Using fine forceps, make horizontal cuts along the jaw. Do this to remove everything about the tongue, leaving behind the brain and skull only 
SECTION: Brain isolation of embryonic and postnatal brains
18. Tilt the head on its side and use forceps to pull away between the skin and the skull to expose the brain. Do this while rotating the head as needed to remove the skin and skull
SECTION: Brain isolation of embryonic and postnatal brains
19. When the brain is exposed, slide forceps along the base of the skull and pinch off the brain to remove it from the head 
SECTION: Processing and Freezing of embryonic and postnatal brains
20. When the brain is isolated, place the brain in 30% Sucrose prepared in 1X PBS 
SECTION: Processing and Freezing of embryonic and postnatal brains
21. Wait until the brain sinks. For embryonic brains, this takes 3-16 hours while postnatal brains take 24-35 hours 
SECTION: Processing and Freezing of embryonic and postnatal brains
22. When the brains have sunk, they are ready to be frozen 
SECTION: Processing and Freezing of embryonic and postnatal brains
23. Fill a bucket with powdered dry ice
SECTION: Processing and Freezing of embryonic and postnatal brains
24. Place Isopentane bottle and the brain’s respective falcon on top of the dry ice. Make sure they are secured within the dry ice 
SECTION: Processing and Freezing of embryonic and postnatal brains
25. Pour the brain into a petri dish 
SECTION: Processing and Freezing of embryonic and postnatal brains
26. Gently scoop the brain from the petri dish and place it on top of a thickly folded paper towel 
SECTION: Processing and Freezing of embryonic and postnatal brains
27. Roll a KimTech Dry wipe and use it to gently roll the brain along the paper towel. This will ensure that the brain is dry 
SECTION: Processing and Freezing of embryonic and postnatal brains
28. Once the brain is dried, slowly drop the brain into the Isopentane solution 
SECTION: Processing and Freezing of embryonic and postnatal brains
29. Leave embryonic brains for 15-20 minutes and postnatal brains for 25-30 minutes in the isopentane bottle for proper freezing 
SECTION: Processing and Freezing of embryonic and postnatal brains
30. Once the freezing is done, gently scoop the brain out of the isopentane bottle and gently run the spoon over a KimTech dry wipe to dry the liquid. Make sure to work quickly in this step, so the brain does not thaw. 
SECTION: Processing and Freezing of embryonic and postnatal brains
31. Transfer the brain into its respective falcon, and place the falcon in -80C. 
SECTION: Processing and Freezing of embryonic and postnatal brains
32. Brains frozen with Isopentane can be stored in the -80C indefinitely.