Apr 19, 2024
Version 2
Mouse Organ Collection (Brain, Bone, Colon, Liver, and Mammary) V.2
- Andrew Davis1,2,
- Aaron Havas1,2,
- Sha Li1,2,
- Laurence Haddadin1,2,
- Diana Jurk3,4,
- Kenneth Kim5,
- Peter Adams1,2
- 1Sanford Burnham Prebys Medical Discovery Institute;
- 2Adams Lab;
- 3Mayo Clinic;
- 4Jurk Lab;
- 5La Jolla Institute of Immunology Histopathology Core
- Andrew Davis: Wrote collected protocol. Source for the Brain isolation;
- Aaron Havas: Source for Liver isolation;
- Sha Li: Source for Bone marrow isolation;
- Laurence Haddadin: Source for the colon isolation;
- Diana Jurk: PI of the Jurk Lab. Provided direction and improvements on bone marrow collection protocol.
- Kenneth Kim: Ken is the director of the histopathology core at LJI. He provided valuable insights and direction on sample preparation for downstream histology, and pointers on the colon collection protocol.
- Peter Adams: PI of Adams Lab and NIH grant awardee
Protocol Citation: Andrew Davis, Aaron Havas, Sha Li, Laurence Haddadin, Diana Jurk, Kenneth Kim, Peter Adams 2024. Mouse Organ Collection (Brain, Bone, Colon, Liver, and Mammary). protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkwx7wl5r/v2Version created by Andrew Davis
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: April 18, 2024
Last Modified: April 19, 2024
Protocol Integer ID: 98429
Keywords: SenNet, Mouse, Bone Marrow, Liver, Brain, Hippocampus, Colon, Mammary
Funders Acknowledgement:
NIH
Grant ID: 1U54AG079758-01
Abstract
This is a mouse dissection protocol intended to collect the 5 organs outlined in the SBPMDI TMC: Brain, Bone Marrow, Colon, Liver, and Mammary. Liver is collected as whole liver as opposed to hepatocytes or perfused liver. Brain is collected alternatively as hippocampus or a whole hemisphere for OCT or FFPE. Bone marrow is collected as viable cells frozen down in freezing media.
Materials
| A | B | C | |
| Name | Catalog number | Source | |
| t-pins | 87T | Avantus | |
| iris scissors | 501758 | World Precision Instruments | |
| forceps | ean code: 0860002672311 | In kit purchased on amazon | |
| cotton tipped swabs or rounded metal bar | ean code: 0860002672311 | In kit purchased on amazon | |
| aluminum dissection tray | 629010 | Carolina Biological Supply | |
| wax pad | DTW-5 | Braintree Scientific | |
| LN2 Dewar + LN2 | 116704B | Fisher | |
| bone scissors | ean code: 0860002672311 | In kit purchased on amazon | |
| OCT molds | 25608-922 | Sakura | |
| OCT | 4583 | Sakura | |
| 1.7ml tubes | 24-282 | Genesee Scientific | |
| RNase-free Ethanol | 3916EA | Decon Labs | |
| Paraformaldehyde | P6148-500G | Sigma-Aldrich | |
| RNase-free water | BP28191 | Fisher Scientific | |
| Refrigerated centrifuge | 5424R | Eppendorf | |
| Cell strainers (70µm) | 22363548 | Fisher | |
| FBS | 35-016-CV | VWR | |
| Mortar and Pestle(s) | B00FGDN8Y6 | Amazon | |
| Metal block | 88-860-105 | Fisher | |
| Ice bucket with metal plate for brain | 07-210-123 | Fisher | |
| RNase-Away or similar | 21402178 (CS) | Fisher | |
| RBC lysis buffer | 555899 | BD biosciences | |
| Histology Cassettes | 18000-130 | VWR | |
| Paraformaldehyde (PFA) | P6148-500G | Sigma Aldrich | |
| Paraformaldehyde (pre-made, optional) | 50980494 | Fisher Scientific (manufacturer is Electron Microscopy Sciences) |
Safety warnings
Always use recommended PPE from your institutional safety department (lab coat, gloves, eye protection, masks, etc.). This can also help to prevent contamination of your samples.
Ethics statement
All experiments using mice should be first approved by the appropriate oversight organization of your locale.
Before start
Make sure to spray down tools and surfaces with RNase-away or similar to avoid RNA degradation of samples.
Pre-collection (at least 1 day before)
Pre-collection (at least 1 day before)
Re-melt and cool wax pad to ensure stable surface for pinning
Prepare labeled plastic cassettes for each PFA tissue and a larger container (250ml+) for depositing the closed cassettes once they have tissues in them. Prepare enough PFA to submerge all cassettes for the collection. Store the made PFA at 4°C, can be made up to a week before dissection date. In a standard 250ml beaker, 150ml should be enough to submerge at least 10 cassettes.
Prepare labeled 50ml conical tubes for bone marrow collection. You will also need a 70µm cell strainer for each of these 50ml conical tubes
Prepare 60ml of wash buffer for each sample for the bone collection. Store at 4°C overnight
Prepare 5ml of RBC lysis buffer for each sample for the bone collection. Can be stored at 4°C over night
Prepare 0.5ml tubes for bone centrifugation (1 for each mouse)
Use a clean pointy metal object (awl, drill, etc), bore a 1-1.5mm hole in the bottom of the 0.5ml tube.
Cut off the cap of the tube at the base of the tube so the lip is nearly perfectly round.
Prepare and label three 2ml freezing tubes for bone marrow collection for each mouse
Prepare and label 1.7ml tubes for tissue collection
Because we are only collecting mammary from female mice, the exact number will also depend on which mice are male or female. (e.g. a male mouse will need 10 labeled tubes, three for bone, three for liver, three for colon, and one for hippocampus, but none for mammary)
Prepare and label OCT cassettes for each tissue for each mouse. The exact number will depend on whether brain is collected as OCT/PFA.
Pre-collection (day of collection)
Pre-collection (day of collection)
Prepare a 10cm dish with 5-10ml dPBS to rinse liver in after excision
Dry-ice buckets with pre-labeled freezer boxes for sample storage
Which go into a -80C freezer after collection is done
Liquid nitrogen dewars with Liquid nitrogen in them for flash freezing tubes
Also need a tool to get the tubes out, we bought a strainer advertised for hot-pot that has a handle canted at roughly 90° to the ladle
Ice buckets with ice in them
at least 1 for the bone team to store bones in 6-well plates in PBS on ice
at least 1 for the brain team to put a metal plate on to do hippocampus isolation on
We use metal tube-racks turned upside down for a flat metal surface in an ice bucket full of ice. This keeps the surface cold while we work on the brain. It occasionally needs to be wiped off to remove condensation, but cleaning the surface between brain isolations should be enough during the procedures.
at least 1 to keep the perfusion media cold
Label paper towels or small pieces of paper to stay with the mouse/dissection pad as it changes hands.
This makes it obvious which mouse is in the tray as it is handed around to different people and reduces the likelihood of mislabeling or forgetting which mouse is where.
Prepare weight boats labeled by mouse and by tissue to transfer tissues from the dissection try to the various teams. This is not a necessary step, but we have found it helpful to keep track of tissues during collections with many mice.
Spray workspace with RNase-away or similar
Wipe down tools with RNase-away or similar
Initial Dissection and Perfusion
Initial Dissection and Perfusion
Set a labeled paper towel or piece of paper down in the tray.
Pin euthanized mouse to wax pad with a t-pin through each paw, with the ventral side of the animal facing away from the wax pad. Try to stretch the paws away from each other during pinning to keep the skin taut.
Gently lift the head of the mouse and use bone scissors to cut the head off at the base of the shoulders. Secondary cuts along remaining skin and ligaments may be necessary to fully separate the head from the body. Give the head to the Brain Team.
Using forceps, pull skin and fur from anterior pelvic region upward away from the body and use scissors to make a ≈1cm cut along the Sagittal plane of the mouse.
Using forceps and scissors, expand the Sagittal cut all the way to the neck of the mouse.
At the inferior end of the Sagittal cut, make a cut along the transverse plane towards the mouse’s right posterior paw (your left), and continue the cut to expose the mammary tissue.
Pull the skin taut and pin to the wax pad, exposing the mammary glands.
Repeat the last step on the mouse’s left side (your right)
Using forceps and scissors, pull peritoneum from anterior pelvic region away from the body and use scissors to make a ≈1cm cut along the transverse plane.
Expand the cut on the peritoneum on both sides laterally and upward toward the ribcage. Pull the peritoneum flap up and away, exposing the abdominal organs.
Whole Body Perfusion
Make a small cut along the diaphragm to allow air into the chest, and continue the cut along the rib cage to expose the heart and lungs. This will help to avoid accidentally puncturing the heart or lungs which can cause problems for the perfusion later.
Make two cuts on the left and right ventral chest through the ribcage using bone scissors to better expose the heart.
Using a 25ga needle, and a 10ml syringe with 10ml of ice-cold dPBS, pierce into the left ventricle of the heart and gently squeeze the plunger to introduce some pressure
Make a cut in to the right auricle of the heart, which will allow blood and PBS to exit the circulatory system at the opposite end of the circulatory system as the perfusion input
Continue to squeeze the plunger to deliver the cold dPBS at 1ml/7s
Using forceps and scissors, follow the intestine down to the pelvis and cut the colon at the most distal you can reach without damaging the surrounding tissue.
Grip the freshly cut end with forceps and pull upward until you can clearly see the caecum. You will feel some pancreas tear away from the colon when you do this.
Make a second cut ≈0.5cm below the caecum, isolating the colon.
Give colon to Colon team
Follow liver Collection steps
Give carcass to Mammary Collection team for mammary removal
Colon Collection
Colon Collection
Receive Colon from Dissection team
Clamp the proximal end of the colon with a set of locking forceps to anchor it, and lean the forceps up against the side of an ice bucket or mouse tray so that the colon hangs down from the locking forceps.
Using forceps and scissors, put one end of the scissors in the colon and cut along the sagittal plane of the colon, making a single flat sheet.
Gently remove any fecal matter still stuck to the now exposed inside of the colon
Make 3 cuts along the colon
From the proximal end (closest to the caecum), cut a 0.5-1cm length and set in an OCT mold into a dry ice + isopropanol bath. Orient the colon vertically in the OCT before adding to the dry ice + isopropanol bath.
From the proximal end, cut another 0.5-1cm length and set in a tube with 4% PFA for 24 hr, then into 70% EtOH
The rest (should be >2cm) should be cut into 2-3 pieces. One piece is set in a tube for RNA/Protein, the rest of the pieces should be spread out along the inside of a 2nd tube to allow for easy retrieval after freezing. Both tubes go into LN2 for flash-freezing.
Liver Collection
Liver Collection
Using a cotton swab or other rounded implement, gently pull the intestinal tract to the side of the mouse, exposing the liver.
Using forceps and scissors, make a cut on the most proximal part of the L-lobe to separate it from the rest of the liver. (The L-lobe is the large, singular lobe on the mouse’s left side (operator's right).)
Orienting the L-lobe from proximal to distal, make 2 cuts through the L-lobe, splitting it into 3 parts.
Most distal portion is cut into smaller pieces (≈3mm2). One piece is set in a tube for RNA/Protein, the rest of the pieces should be spread out along the inside of a 2nd tube to allow for easy retrieval after freezing. Both tubes go into LN2 for flash-freezing.
The middle portion is put in OCT and then dry-ice + isopropanol bath. Orient in OCT with one of the two cuts facing the bottom of the mold
Proximal portion is put in 4% PFA for H&E and RNAScope
Collect the other lobes in separate tubes to have more tissue for other analyses
Mammary Collection
Mammary Collection
Remove the lymph node from the mammary glands
Using forceps and scissors, pull on the exposed mammary glands and use the scissors to cut the mammary glands away from the skin.
Cut the (mouse's) left mammary gland into 2 even pieces
One piece goes in an OCT mold into a dry ice + isopropanol bath
One piece goes into a tube with 4% PFA for 24 hr, then into 70% EtOH
The (mouse's) right mammary gland should be cut up into smaller pieces (≈3mm2). One piece is set in a tube for RNA/Protein, the rest of the pieces should be spread out along the inside of a 2nd tube to allow for easy retreival after freezing. Both tubes go into LN2 for flash-freezing.
Give carcass to Bone collection team to remove the leg bones
Brain Collection
Brain Collection
Receive the Head from the Dissection team, or follow Mouse Dissection protocol steps 1 and 2
Hold head in one hand and pull the skin away from the posterior of the head and towards the anterior of the head to reveal the skull; if necessary, make longitudinal cuts along the skin with scissors to make the skin easier to remove.
It is recommended to make two cuts under the skin where the ears meet the skull to separate the skin of the ear canal that connects to the skull. This will allow you to pull the skin further back and more easily expose the skull.
Once the skull is exposed, take small scissors and cut the skull following the superior midline of the brain so as to avoid damaging the brain tissue as much as possible.
The skull may fracture during this process, carefully remove any loose pieces and continue
Using fine forceps or your fingers, grip the cut edge of the skull nearest the cut and pull the pieces up until they fracture; remove the fractured pieces and repeat until the brain is exposed (you will need an open area roughly 1-1.5cm2 to get the brain out of the skull).
Using a spatula or tweezers: slip a tool underneath the brain between the brain and skull to sever the occular nerves connecting the brain to the eyes.
Use a spatula to gently pull the brain out of the skull. There may be remaining neural tissue connections to the spinal cord or eyes that you will need to sever.
Split brain along the sagittal plane using a razor blade or scalpel.
Continue to Hippocampus isolation or OCT/FFPE preparation
Options: Because there are 3 desired collection formats and only 2 halves of the brain, you may need to make decisions between taking more FFPE, OCT, or isolated hippocampus, or taking different formats from alternate mice, as suits your downstream plans. For example, for some experiments, we have taken isolated hippocampus from both hemispheres of every other mouse to ensure we have enough hippocampus tissue for snSeq.
Hippocampus isolation for snRNA-seq
Hippocampus isolation for snRNA-seq
Take the left half of the brain and use a scalpel or razor blade to remove the olfactory bulb
Make a cut along the midbrain to remove the cerebellum, pons and medulla.
Using curved forceps and/or small spatulas, gently remove the thalamus and hypothalamus from the cortex and hippocampus, exposing the hippocampus.
Use a spatula to isolate the hippocampus from the cortex and put in a tube and set in LN2
Choose to collect OCT or FFPE for each mouse. Ideally your n is large enough to have at least 3 for each.
Collect OCT32 steps
Take the right half of the brain and set in an OCT mold and set in a dry ice + isopropanol bath
Bone Marrow Collection
Bone Marrow Collection
Receive carcass from Mammary Collection team
Remove legs at the pelvis
Using forceps and scissors, remove the bulk of tissue from the leg bones
Using gloved hands and a lint-free cloth, remove as much remaining muscle and ligaments as possible.
Cut the right femur in the middle and notch the proximal end to allow better diffusion of PFA into the tissue. Set in 4% PFA for 24hr, then into 70% EtOH.
Set remaining bones in pre-prepared wash buffer (2% FBS in PBS) on ice
Setup an ice bucket with a labeled 6-well plate in it with wash buffer in each of the wells ahead of time
Repeat until all bones from the day’s experiment are collected
Bone marrow recovery from bones in ice cold PBS gives good yield for at least 5 hours after mouse death (longer intervals have not been tested, sooner is always preferred if possible)
After all of the bones are collected:
Using a scalpel, make cuts at the metaphyses of the remaining bones (1.5 femurs and 2 tibia)
This may require some sawing with a scalpel, and doing this on a wax pad has been the most effective for me. Making these cuts will turn the bones into tubes open at either end.
Add the bones to a 0.5ml tube with a pre-punched hole in the bottom
To help avoid more shear forces on the cells, I try to put the bones in with the larger hole facing downward toward the bottom of the tube. For the tibia, it is the proximal end; for the femurs, it usually is the same size on either end
insert the 0.5ml tube into a labeled 1.7ml tube
Weigh the 1.7ml tubes and make counterbalances if the weights are off from each other more than ≈20%
Spin the cells down at 3000xg for 1min at 4°C
Open the labeled 50 ml conicals and put 70µl cell strainers on them
Use a 1000µl pipet to add 1ml of wash buffer to the tube and pipet up and down to mix
Use a 1000µl pipet to take the resuspended cells from the 1.7ml tube, and run them through a cell strainer into a labeled 50ml conical tube.
Run 39ml of wash buffer through the top of the cell strainer to help collect any remaining cells in the strainer.
Remove strainers and put cap on 50ml conical tube
Spin cells down (All spins at 500 x g for 3 min a 4°C unless otherwise noted)
Decant supernatant without distrubing the pellet and then add 5ml RBC lysis buffer
Allow reaction to proceed for 4 minutes
Add 10ml of wash buffer to dilute the reaction and spin for 500xg for 3min at 4°C
Decant supernatant without disturbing the pellet
Add 10ml of wash buffer
Spin down (500xg, 3min, 4°C)
Decant supernatant without disturbing the pellet
Resuspend the pellet in 1ml freezing media (10% DMSO in FBS)
Distribute cells into 3x 2ml cryo tube (≈330µl each) in a slow-freeze container at -80°C