TITLE: Mouse brain, gut  and plasma collection
AUTHORS: Livia Hecke Morais

[DESCRIPTION]
Protocol used in the Mazmanian lab for collecting brain and gut tissues and plasma from mouse for metabolomics. 

Note that any protocol involving animals should be approved by your Institutional Animal Care
and Use Committee (IACUC) before use.

[STEPS]
SECTION: Mouse brain, gut and plasma collection
1. Euthanize the mouse inside a fume hood by decapitation using scissors (or by alternative method approved by your IACUC).
SECTION: Mouse brain, gut and plasma collection
2. Use straight scissors to separate the head from the rest of the body.
SECTION: Mouse brain, gut and plasma collection
7. Use a single-edge razor blade to section the brain tissue in slices of approximately  . 
SECTION: Mouse brain, gut and plasma collection
8. Dissect brain regions of interest according to a reference brain atlas.

 

SECTION: Mouse brain, gut and plasma collection
26. Store samples at -80 °C until use.

SECTION: Mouse brain, gut and plasma collection
27. For plasma samples
SECTION: Mouse brain, gut and plasma collection
29. Separate plasma by centrifugation at 2.500 x g for 10 min

SECTION: Mouse brain, gut and plasma collection
30. Transfer plasma to a pre-cooled  
SECTION: Mouse brain, gut and plasma collection
6. Place the brain in an ice-chilled stainless steel coronal matrix. 
SECTION: Mouse brain, gut and plasma collection
3. For brain samples
SECTION: Mouse brain, gut and plasma collection
13. For gut samples 
SECTION: Mouse brain, gut and plasma collection
18. Remove the intestine from the duodenum to the rectum. 
SECTION: Mouse brain, gut and plasma collection
16. Insert the sterilized scissors below the skin to expose the abdominal muscle.  
SECTION: Mouse brain, gut and plasma collection
17. Make another incision through the abdominal muscle to access the internal organs.
SECTION: Mouse brain, gut and plasma collection
5. Use forceps, a Boehler bone cutter, and serrated forceps to detach any residual muscle tissue from the posterior and inferior portions of the skull. 
SECTION: Mouse brain, gut and plasma collection
9. Weigh samples and record for future reference.
SECTION: Mouse brain, gut and plasma collection
14. Using large scissors and large forceps, make an incision through the abdominal skin.
SECTION: Mouse brain, gut and plasma collection
4. Use a surgical blade or straight scissors to rapidly remove the brain from the skull by severing the proximal end of the spinal cord from the occipital bone of the skull
SECTION: Mouse brain, gut and plasma collection
28. At Room temperature collect trunk blood in   

SECTION: Mouse brain, gut and plasma collection
10. Add samples to   

SECTION: Mouse brain, gut and plasma collection
11. Snap-freeze on dry ice.
SECTION: Mouse brain, gut and plasma collection
12. Store samples at -80 °C until use.

SECTION: Mouse brain, gut and plasma collection
15. Sterilize scissors by spraying with 70 % (v/v) EtOH.

SECTION: Mouse brain, gut and plasma collection
19. On an ice-chilled dissection tray, dissect desired intestinal samples.

 

SECTION: Mouse brain, gut and plasma collection
20. To sample contents of small intestine, cecum, and distal colon, remove contents using a 5 mL syringe and 14 G needle.
SECTION: Mouse brain, gut and plasma collection
21. Cut tissue samples into   sections.

SECTION: Mouse brain, gut and plasma collection
22. Weigh samples and record for future reference.
SECTION: Mouse brain, gut and plasma collection
23. Add tissue samples to   

SECTION: Mouse brain, gut and plasma collection
25. Snap-freeze samples on dry ice.
SECTION: Mouse brain, gut and plasma collection
24. Add tissue content samples to sterile   

SECTION: Mouse brain, gut and plasma collection
31. Store at -80 °C until use.