TITLE: Mouse Organ Collection (Brain, Bone, Colon, Liver, and Mammary)
AUTHORS: Andrew Davis, Aaron Havas, Sha Li, Laurence Haddadin, Diana Jurk, Kenneth Kim, Peter Adams

[DESCRIPTION]
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.

[BEFORE_START]
Make sure to spray down tools and surfaces with RNase-away or similar to avoid RNA degradation of samples.

[STEPS]
SECTION: Pre-collection (at least 1 day before)
1. Re-melt and cool wax pad to ensure stable surface for pinning
SECTION: Pre-collection (at least 1 day before)
2. 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.
SECTION: Pre-collection (at least 1 day before)
3. Prepare labeled 50ml conical tubes for bone marrow collection.  You will also need a 70µm cell strainer for each of these 50ml conical tubes
SECTION: Pre-collection (at least 1 day before)
4. Prepare 60ml of wash buffer for each sample for the bone collection.  Store at 4°C overnight
SECTION: Pre-collection (at least 1 day before)
5. Prepare 5ml of RBC lysis buffer for each sample for the bone collection.  Can be stored at 4°C over night
SECTION: Pre-collection (at least 1 day before)
6. Prepare 0.5ml tubes for bone centrifugation (1 for each mouse)
SECTION: Pre-collection (at least 1 day before)
6.1. Use a clean pointy metal object (awl, drill, etc), bore a 1-1.5mm hole in the bottom of the 0.5ml tube.
SECTION: Pre-collection (at least 1 day before)
6.2. Cut off the cap of the tube at the base of the tube so the lip is nearly perfectly round.
SECTION: Pre-collection (at least 1 day before)
7. Prepare and label three 2ml freezing tubes for bone marrow collection for each mouse
SECTION: Pre-collection (at least 1 day before)
8. Prepare and label 1.7ml tubes for tissue collection
SECTION: Pre-collection (at least 1 day before)
8.1. 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)
SECTION: Pre-collection (at least 1 day before)
9. Prepare and label OCT cassettes for each tissue for each mouse.  The exact number will depend on whether brain is collected as OCT/PFA.
SECTION: Pre-collection (day of collection)
10. Prepare a 10cm dish with 5-10ml dPBS to rinse liver in after excision
SECTION: Pre-collection (day of collection)
11. Dry-ice buckets with pre-labeled freezer boxes for sample storage
SECTION: Pre-collection (day of collection)
11.1. Which go into a -80C freezer after collection is done
SECTION: Pre-collection (day of collection)
12. Liquid nitrogen dewars with Liquid nitrogen in them for flash freezing tubes
SECTION: Pre-collection (day of collection)
12.1. 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
SECTION: Pre-collection (day of collection)
13. Ice buckets with ice in them
SECTION: Pre-collection (day of collection)
13.1. at least 1 for the bone team to store bones in 6-well plates in PBS on ice
SECTION: Pre-collection (day of collection)
13.2. at least 1 for the brain team to put a metal plate on to do hippocampus isolation on
SECTION: Pre-collection (day of collection)
13.3. 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.
SECTION: Pre-collection (day of collection)
14. Label paper towels or small pieces of paper to stay with the mouse/dissection pad as it changes hands.
SECTION: Pre-collection (day of collection)
14.1. 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.
SECTION: Pre-collection (day of collection)
16. Spray workspace with RNase-away or similar
SECTION: Pre-collection (day of collection)
17. Wipe down tools with RNase-away or similar
SECTION: Initial Dissection and Perfusion
18. Set a labeled paper towel or piece of paper down in the tray.
SECTION: Initial Dissection and Perfusion
19. 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.
SECTION: Initial Dissection and Perfusion
20. 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.
SECTION: Initial Dissection and Perfusion
21. 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.
SECTION: Initial Dissection and Perfusion
22. Using forceps and scissors, expand the Sagittal cut all the way to the neck of the mouse.
SECTION: Initial Dissection and Perfusion
23. 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.
SECTION: Initial Dissection and Perfusion
24. Pull the skin taut and pin to the wax pad, exposing the mammary glands.
SECTION: Initial Dissection and Perfusion
25. Repeat the last step on the mouse’s left side (your right)
SECTION: Initial Dissection and Perfusion
26. 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.
SECTION: Initial Dissection and Perfusion
27. 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.
SECTION: Initial Dissection and Perfusion
29. 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.
SECTION: Initial Dissection and Perfusion
32. Give colon to Colon team
SECTION: Initial Dissection and Perfusion
33. Follow liver Collection steps
SECTION: Initial Dissection and Perfusion
34. Give carcass to Mammary Collection team for mammary removal
SECTION: Colon Collection
35. Receive Colon from Dissection team
SECTION: Initial Dissection and Perfusion
30. 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.
SECTION: Initial Dissection and Perfusion
31. Make a second cut ≈0.5cm below the caecum, isolating the colon.
SECTION: Colon Collection
36. 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.
SECTION: Colon Collection
37. 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.
SECTION: Colon Collection
38. Gently remove any fecal matter still stuck to the now exposed inside of the colon
SECTION: Colon Collection
39. Make 3 cuts along the colon
SECTION: Colon Collection
39.1. 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.
SECTION: Colon Collection
39.2. 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
SECTION: Colon Collection
39.3. 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.
SECTION: Liver Collection
40. Using a cotton swab or other rounded implement, gently pull the intestinal tract to the side of the mouse, exposing the liver.
SECTION: Liver Collection
41. 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).)
SECTION: Liver Collection
42. Orienting the L-lobe from proximal to distal, make 2 cuts through the L-lobe, splitting it into 3 parts.
SECTION: Liver Collection
42.1. 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.
SECTION: Liver Collection
42.2. 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
SECTION: Liver Collection
42.3. Proximal portion is put in 4% PFA for H&E and RNAScope
SECTION: Mammary Collection
44. Remove the lymph node from the mammary glands
SECTION: Mammary Collection
45. Using forceps and scissors, pull on the exposed mammary glands and use the scissors to cut the mammary glands away from the skin.
SECTION: Mammary Collection
46. Cut the (mouse's) left mammary gland into 2 even pieces
SECTION: Mammary Collection
46.1. One piece goes in an OCT mold into a dry ice + isopropanol bath
SECTION: Mammary Collection
46.2. One piece goes into a tube with 4% PFA for 24 hr, then into 70% EtOH
SECTION: Mammary Collection
47. 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.
SECTION: Mammary Collection
48. Give carcass to Bone collection team to remove the leg bones
SECTION: Brain Collection
49. Receive the Head from the Dissection team, or follow Mouse Dissection protocol steps 1 and 2
SECTION: Brain Collection
50. 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.
SECTION: Brain Collection
50.1. 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.
SECTION: Brain Collection
51. 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.
SECTION: Brain Collection
52. The skull may fracture during this process, carefully remove any loose pieces and continue
SECTION: Brain Collection
53. 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).
SECTION: Brain Collection
54. 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.
SECTION: Brain Collection
55. 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.
SECTION: Brain Collection
56. Split brain along the sagittal plane using a razor blade or scalpel.
SECTION: Brain Collection
57. Continue to Hippocampus isolation or OCT/FFPE preparation
SECTION: Hippocampus isolation for snRNA-seq
58. Take the left half of the brain and use a scalpel or razor blade to remove the olfactory bulb
SECTION: Hippocampus isolation for snRNA-seq
59. Make a cut along the midbrain to remove the cerebellum, pons and medulla.
SECTION: Hippocampus isolation for snRNA-seq
60. Using curved forceps and/or small spatulas, gently remove the thalamus and hypothalamus from the cortex and hippocampus, exposing the hippocampus.
SECTION: Hippocampus isolation for snRNA-seq
61. Use a spatula to isolate the hippocampus from the cortex and put in a tube and set in LN2
SECTION: Brain for OCT/FFPE preparation (One or the other)
62. Choose to collect OCT or FFPE for each mouse.  Ideally your n is large enough to have at least 3 for each.
SECTION: Bone Marrow Collection
63. Receive carcass from Mammary Collection team
SECTION: Bone Marrow Collection
64. Remove legs at the pelvis
SECTION: Bone Marrow Collection
65. Using forceps and scissors, remove the bulk of tissue from the leg bones
SECTION: Bone Marrow Collection
66. Using gloved hands and a lint-free cloth, remove as much remaining muscle and ligaments as possible.
SECTION: Bone Marrow Collection
67. Cut the right femur in the middle and notch the proximal end.  Set in 4% PFA for 24hr, then into 70% EtOH.
SECTION: Bone Marrow Collection
68. Set remaining bones in pre-prepared wash buffer (2% FBS in PBS) on ice
SECTION: Bone Marrow Collection
68.1. Setup an ice bucket with a labeled 6-well plate in it with wash buffer in each of the wells ahead of time
SECTION: Bone Marrow Collection
69. Repeat until all bones from the day’s experiment are collected
SECTION: Bone Marrow Collection
70. After all of the bones are collected:
SECTION: Bone Marrow Collection
71. Using a scalpel, make cuts at the metaphyses of the remaining bones (1.5 femurs and 2 tibia)
SECTION: Bone Marrow Collection
71.1. This may require some sawing, 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.
SECTION: Bone Marrow Collection
72. Add the bones to a 0.5ml tube with a pre-punched hole in the bottom
SECTION: Bone Marrow Collection
72.1. 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
SECTION: Bone Marrow Collection
73. Add the 0.5ml tube to a labeled 1.7ml tube
SECTION: Bone Marrow Collection
74. Weigh the 1.7ml tubes and make counterbalances if the weights are off from each other more than ≈20%
SECTION: Bone Marrow Collection
75. Spin the cells down at 3000xg for 1min at 4°C
SECTION: Bone Marrow Collection
76. Open the labeled 50 ml conicals and put 70µl cell strainers on them
SECTION: Bone Marrow Collection
77. Use a 1000µl pipet to add 1ml of wash buffer to the tube and pipet up and down to mix
SECTION: Bone Marrow Collection
78. 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.
SECTION: Bone Marrow Collection
79. Run 39ml of wash buffer through the top of the cell strainer to help collect any remaining cells in the strainer.
SECTION: Bone Marrow Collection
80. Remove strainers and put cap on 50ml conical tube
SECTION: Bone Marrow Collection
81. Spin cells down (All spins at 500 x g for 3 min a 4°C unless otherwise noted)
SECTION: Bone Marrow Collection
82. Decant supernatant without disturbing the pellet and then add 5ml RBC lysis buffer
SECTION: Bone Marrow Collection
83. Allow reaction to proceed for 4 minutes
SECTION: Bone Marrow Collection
84. Add 10ml of wash buffer to dilute the reaction and spin for 500xg for 3min at 4°C
SECTION: Bone Marrow Collection
85. Decant supernatant without disturbing the pellet
SECTION: Bone Marrow Collection
86. Add 10ml of wash buffer
SECTION: Bone Marrow Collection
87. Spin down (500xg, 3min, 4°C)
SECTION: Bone Marrow Collection
88. Decant supernatant without disturbing the pellet
SECTION: Bone Marrow Collection
89. Resuspend the pellet in 1ml freezing media (10% DMSO in FBS)
SECTION: Bone Marrow Collection
90. Distribute cells into 3x 2ml cryo tube (≈330µl each) in a slow-freeze container at -80°C
SECTION: Bone Marrow Collection
63. Receive carcass from Mammary Collection team
SECTION: Bone Marrow Collection
64. Remove legs at the pelvis
SECTION: Bone Marrow Collection
65. Using forceps and scissors, remove the bulk of tissue from the leg bones
SECTION: Bone Marrow Collection
66. Using gloved hands and a lint-free cloth, remove as much remaining muscle and ligaments as possible.
SECTION: Bone Marrow Collection
67. 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.
SECTION: Bone Marrow Collection
68. Set remaining bones in pre-prepared wash buffer (2% FBS in PBS) on ice
SECTION: Bone Marrow Collection
68.1. Setup an ice bucket with a labeled 6-well plate in it with wash buffer in each of the wells ahead of time
SECTION: Bone Marrow Collection
69. Repeat until all bones from the day’s experiment are collected
SECTION: Bone Marrow Collection
70. After all of the bones are collected:
SECTION: Bone Marrow Collection
71. Using a scalpel, make cuts at the metaphyses of the remaining bones (1.5 femurs and 2 tibia)
SECTION: Bone Marrow Collection
71.1. 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.
SECTION: Bone Marrow Collection
72. Add the bones to a 0.5ml tube with a pre-punched hole in the bottom
SECTION: Bone Marrow Collection
72.1. 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
SECTION: Bone Marrow Collection
73. insert the 0.5ml tube into a labeled 1.7ml tube
SECTION: Bone Marrow Collection
74. Weigh the 1.7ml tubes and make counterbalances if the weights are off from each other more than ≈20%
SECTION: Bone Marrow Collection
75. Spin the cells down at 3000xg for 1min at 4°C
SECTION: Bone Marrow Collection
76. Open the labeled 50 ml conicals and put 70µl cell strainers on them
SECTION: Bone Marrow Collection
77. Use a 1000µl pipet to add 1ml of wash buffer to the tube and pipet up and down to mix
SECTION: Bone Marrow Collection
78. 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.
SECTION: Bone Marrow Collection
79. Run 39ml of wash buffer through the top of the cell strainer to help collect any remaining cells in the strainer.
SECTION: Bone Marrow Collection
80. Remove strainers and put cap on 50ml conical tube
SECTION: Bone Marrow Collection
81. Spin cells down (All spins at 500 x g for 3 min a 4°C unless otherwise noted)
SECTION: Bone Marrow Collection
82. Decant supernatant without distrubing the pellet and then add 5ml RBC lysis buffer
SECTION: Bone Marrow Collection
83. Allow reaction to proceed for 4 minutes
SECTION: Bone Marrow Collection
84. Add 10ml of wash buffer to dilute the reaction and spin for 500xg for 3min at 4°C
SECTION: Bone Marrow Collection
85. Decant supernatant without disturbing the pellet
SECTION: Bone Marrow Collection
86. Add 10ml of wash buffer
SECTION: Bone Marrow Collection
87. Spin down (500xg, 3min, 4°C)
SECTION: Bone Marrow Collection
88. Decant supernatant without disturbing the pellet
SECTION: Bone Marrow Collection
89. Resuspend the pellet in 1ml freezing media (10% DMSO in FBS)
SECTION: Bone Marrow Collection
90. Distribute cells into 3x 2ml cryo tube (≈330µl each) in a slow-freeze container at -80°C
SECTION: Pre-collection (day of collection)
15. 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. 
SECTION: Pre-collection (day of collection)
13.4. at least 1 to keep the perfusion media cold
SECTION: Initial Dissection and Perfusion
28. Whole Body Perfusion
SECTION: Initial Dissection and Perfusion
28.1. 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.
SECTION: Initial Dissection and Perfusion
28.2. Make two cuts on the left and right ventral chest through the ribcage using bone scissors to better expose the heart.
SECTION: Initial Dissection and Perfusion
28.5. Continue to squeeze the plunger to deliver the cold dPBS at 1ml/7s
SECTION: Initial Dissection and Perfusion
28.4. 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
SECTION: Initial Dissection and Perfusion
28.3. 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
SECTION: Liver Collection
43. Collect the other lobes in separate tubes to have more tissue for other analyses
SECTION: Brain Collection
57.1. 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.
SECTION: Bone Marrow Collection
69.1. 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)