Sep 26, 2024
Version 3
Mouse brain, gut and plasma collection V.3
- 1California Institute of Technology
Protocol Citation: Livia Hecke Morais 2024. Mouse brain, gut and plasma collection. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn3pkzl5d/v3Version created by Catherine Oikonomou
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 13, 2024
Last Modified: September 26, 2024
Protocol Integer ID: 108401
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-020495
Disclaimer
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
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.
Protocol materials
Precellys 2mL Soft Tissue Homogenizing Ceramic BeadsCayman Chemical CompanyCatalog #10011152
In 2 steps
Safe-Lock Tubes 2 ml Biopur (preferably packed individually)EppendorfCatalog #0030121597
Step 24
Vacutainer Hemogard Closure Plastic K3-EDTA TubeBD BiosciencesCatalog #367836
Step 28
Thermo Scientific Pierce Microcentrifuge Tubes, 1.5 mLThermo Fisher ScientificCatalog #69715
Step 30
Mouse brain, gut and plasma collection
Mouse brain, gut and plasma collection
10m
10m
Euthanize the mouse inside a fume hood by decapitation using scissors (or by alternative method approved by your IACUC).
Use straight scissors to separate the head from the rest of the body.
For brain samples
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
Use forceps, a Boehler bone cutter, and serrated forceps to detach any residual muscle tissue from the posterior and inferior portions of the skull.
Place the brain in an ice-chilled stainless steel coronal matrix.
Use a single-edge razor blade to section the brain tissue in slices of approximately 1 mm .
Dissect brain regions of interest according to a reference brain atlas.
Note
For striatum and motor cortex, we use slices from +1.54 mm to +0.10 mm anterior-to-posterior relative to bregma. For substantia nigra, we use slices from -2.70 mm to -3.52 mm relative to bregma. For brainstem, we start at the superior colliculus of the midbrain and cut obliquely to meet the pituitary gland.
Weigh samples and record for future reference.
Add samples to Precellys 2mL Soft Tissue Homogenizing Ceramic BeadsCayman Chemical CompanyCatalog #10011152
Snap-freeze on dry ice.
Store samples at -80 °C until use.
For gut samples
Using large scissors and large forceps, make an incision through the abdominal skin.
Sterilize scissors by spraying with 70 % (v/v) EtOH.
Insert the sterilized scissors below the skin to expose the abdominal muscle.
Make another incision through the abdominal muscle to access the internal organs.
Remove the intestine from the duodenum to the rectum.
On an ice-chilled dissection tray, dissect desired intestinal samples.
Note
For the small intestine, we dissect from 1 cm distal to the stomach to 1 cm proximal to the cecum. For duodenum, we dissect from 2 cm distal to the pyloric sphincter. For ileum, we take from 2 cm proximal to the cecum. For large intestine, we dissect from distal of the cecum to 10 mm proximal to the anus. For distal colon, we dissect 1 cm proximal to the rectum.
To sample contents of small intestine, cecum, and distal colon, remove contents using a 5 mL syringe and 14 G needle.
Cut tissue samples into 1 cm sections.
Weigh samples and record for future reference.
Add tissue samples to Precellys 2mL Soft Tissue Homogenizing Ceramic BeadsCayman Chemical CompanyCatalog #10011152
Add tissue content samples to sterile Safe-Lock Tubes 2 ml Biopur (preferably packed individually)EppendorfCatalog #0030121597
Snap-freeze samples on dry ice.
Store samples at -80 °C until use.
For plasma samples
At Room temperature collect trunk blood in Vacutainer Hemogard Closure Plastic K3-EDTA TubeBD BiosciencesCatalog #367836
Separate plasma by centrifugation at 2.500 x g for 00:10:00
10m
Transfer plasma to a pre-cooled Thermo Scientific Pierce Microcentrifuge Tubes, 1.5 mLThermo Fisher ScientificCatalog #69715
Store at -80 °C until use.