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. Protocol has been approved by the California Institute of Technology’s Institutional Animal Care
and Use Committee (IACUC).

[STEPS]
SECTION: Mouse brain, gut and plasma collection
1. At 4 months of age, mice is euthanized inside chemical hood by decapitation using scissors (as approved by IACUC protocol)
SECTION: Mouse brain, gut and plasma collection
2. The head is separated from the rest of the body for brain and gut dissections using straight scissors 
SECTION: Mouse brain, gut and plasma collection
7. Brain tissue is sectioned in slices of approximately 1 mm using single edge razor blades. 
SECTION: Mouse brain, gut and plasma collection
8. Substantia nigra, striatum, motor cortex(referred to as cortex) and caudal brainstem (referred to as brainstem) were dissected within three minutes using a brain atlas as reference1 . For striatal brain slices spanned from anterior to posterior (AP) +1.54 mm to +0.10 mm relative to bregma. Motor cortex was taken from same slices as to striatum. Substantia nigra slices were taken from anterior to posterior (AP) -2.70 mm to -3.52 mm relative to bregma.  Brainstem was dissected starting at superior colliculus of idbrain and cut obliquely to meet the pituitary gland.
SECTION: Mouse brain, gut and plasma collection
23. All tissue samples were weighed, and stored at Precellys‱ 2 mL Soft Tissue Homogenizing Ceramic Beads tubes (Catalog number: 10011152), snap-frozen in dry ice and stored at -80ºC until processing. Tissue contents were stored in sterile 2.0mL Safe-Lock Microcentrifuge tube.

SECTION: Mouse brain, gut and plasma collection
24. Trunk blood collection and plasma
SECTION: Mouse brain, gut and plasma collection
26. Plasma was separated by centrifugation at 2,500 x g for 10 minutes 
SECTION: Mouse brain, gut and plasma collection
27. Plasma was transferred to a pre-cooled collection vial Thermo Scientific Pierce 1.5 mL Capacity Microcentrifuge Tubes (Catalog number: 69715) and stored at -80ºC until processing
SECTION: Mouse brain, gut and plasma collection
6. Brain is placed in an ice-chilled stainless steel coronal matrix. 
SECTION: Mouse brain, gut and plasma collection
3. For brain dissection
SECTION: Mouse brain, gut and plasma collection
11. Gut samples collection
SECTION: Mouse brain, gut and plasma collection
15. The intestine from duodenum to rectum is removed. 
SECTION: Mouse brain, gut and plasma collection
17. The small intestine is separated by cutting 1 cm distal to stomach and until 1 cm proximal to cecum. 
Duodenum samples were taken from 2cm distal to pyloric sphincter. Ileum is taken 2 cm proximal to cecum. 
SECTION: Mouse brain, gut and plasma collection
13. Next, sprayed scissors with 70% EtOH is inserted below skin to expose the abdominal muscle.  
SECTION: Mouse brain, gut and plasma collection
14. Another incision is made into the abdomen muscle to access internal organs.
SECTION: Mouse brain, gut and plasma collection
5. 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
20. Large intestine was taken by cutting distal to cecum and 10 mm proximal to anus. 
SECTION: Mouse brain, gut and plasma collection
19. Cecum is separated and cecum content is removed using a 5 mL syringe and 14 G needle 
SECTION: Mouse brain, gut and plasma collection
9. All tissue samples were weighed, stored in and stored at Precellys‱ 2 mL Soft Tissue Homogenizing Ceramic Beads tubes (Catalog number: 10011152), snap-frozen in dry ice and stored at -80ºC
until processing.
SECTION: Mouse brain, gut and plasma collection
16. All intestinal tissue samples were sized to 1 cm
SECTION: Mouse brain, gut and plasma collection
18. Small intestine content is removed using a 5 mL syringe and 14 G needle 
SECTION: Mouse brain, gut and plasma collection
12. Using large scissors and large forceps, an incision is made into abdominal skin
SECTION: Mouse brain, gut and plasma collection
4.  The brain is rapidly removed from the skull by serving the proximal end of the neck and vertebrae from the occipital bone of the skull using a surgical blade or straight scissors. 
SECTION: Mouse brain, gut and plasma collection
21. Only distal colon was taken by cutting the portion 1 cm proximal to the rectum.


SECTION: Mouse brain, gut and plasma collection
25. After Trunk blood was collected in BD Vacutainer‱ Hemogard‱ Closure Plastic K3-Edta Tubes (Catalog number: 15349700) and kept at room temperature before plasma separation.

SECTION: Mouse brain, gut and plasma collection
22. Distal colon content was removed using a 5 mL syringe and 14 G needle 
SECTION: Mouse brain, gut and plasma collection
10. References: 
1. Paxinos, G. and Franklin, K.B.J. (2001) The Mouse Brain in Stereotaxic Coordinates. 2nd Edition, Academic Press, San Diego.