TITLE: Preparation and shipping of mouse tibiae for multiphoton microscopy
AUTHORS: Chad A Lerner, Sofiya Galkina, Gen Matsumae, Simon Melov, Tamara Alliston, Akos A Gerencser

[DESCRIPTION]
Isolation and packaging of mouse tibiae for overnight shipment for bioenergetic analysis of live in situ osteocytes. 

[STEPS]
SECTION: Consumables and Tools
2. Dissection tools and surfaces should be clean, free of detergents, alcohol or disinfectants. 
Tool cleaning:
Detergent wash
Alcohol wash (ethanol or isopropanol)
Water
PBS
SECTION: Cleaning of bones - lab bench
13. Place a 60mm dish on a dissection microscope and half-fill it with room temperature nutrient medium and transfer one bone with tweezer into it.
 


SECTION: Packaging for shipping
22. Seal the transport plate
Make sure that the top of the transport plate is dry. 
Peal backing of breathable film
Holding at the edges attach the film on the plate. Massage the film that it attaches evenly in between wells
Secure the lid on two sides with lab tape, not obstructing air gaps.
  

SECTION: Consumables and Tools
3. Label one 2ml tube for each bone and fill with 1.8ml of nutrient medium (Keep the bottle of nutrient medium sterile. Open it in the hood only). 
Leave the tubes uncapped, but covered with a small square of self-adhesive membrane (cut one breathable membrane up with scissors).
Keep the tubes at RT.
Fill two 50ml tubes with nutrient medium. This can be used on the bench to fill the 60ml dishes and the syringe when cleaning the bones.
SECTION: Consumables and Tools
4. In a 15ml conical supplement nutrient medium to make transport medium:
Measure 40mg BSA (0.4 % w/v final)
add 10ml nutrient medium
add 100µl ITS
add 100µl penicillin/streptomycin/amphotericin B 
Sterile filter using a syringe and syringe filter
SECTION: Dissection of mice - vivarium
5. Mouse is euthanized in CO2 chamber according to the IACUC approved protocol.

 

SECTION: Dissection of mice - vivarium
6. Mouse hindlimbs are rinsed with PBS, fixed to a dissection board with pins. Do not use alcohol or disinfectants.
SECTION: Dissection of mice - vivarium
7. Cut through the entire hind limb and expose the muscles underneath with the help of scissors.
SECTION: Dissection of mice - vivarium
8. Carefully remove the skin as well as any hair or fur.
SECTION: Dissection of mice - vivarium
9. Coarsely remove muscles without damaging bones by holding and cutting corresponding tendons.
SECTION: Dissection of mice - vivarium
10. Expose tibia and fibula and cut them through knee and ankle joints. Keep tibia and fibula together for now. Take extra care not to hold the tibia on the surface that is going to be analyzed, indicated in the picture by the ellipse (flat part of the shaft between crest and fibula stump:
 
SECTION: Dissection of mice - vivarium
11. Transfer bone specimens into the tube with transport medium. Do not cap the tubes, just replace the membrane. Keep tubes at room temperature (do not use ice). Samples should wait here for completion of all dissection.
SECTION: Cleaning of bones - lab bench
14. With a small scissors cut off fibula proximally:
Grab on the fibula that will be discarded (hold vertically knee pointing upwards; in terms of microscope view field)
Insert open scissors vertically between tibia and fibula, cutting upwards
Cut off fibula by cutting through knee joint and remaining muscles in the way
SECTION: Cleaning of bones - lab bench
15. With a small scissors cut off the fibula distally:
Insert open scissors vertically between tibia and fibula, cutting downwards
Cut off fibula leaving only a small ~1mm stomp
SECTION: Cleaning of bones - lab bench
16. Cut off the knee joint
Hold tibia at the distal end, below the fibula stump with tweezers
Hold tibia perpendicular to the scissors, in an angle that you will cut in the plane of the crest, so cutting it rather than crushing it
Cut as close as possible to the knee joint
The cut should expose marrow.
SECTION: Cleaning of bones - lab bench
17. Cut distal end ~2mm below the fibula stump
Hold tibia at the level of the fibula stump
Cut below, carefully not to crack.
The cut should expose big enough hole so the marrow can be blown out
SECTION: Cleaning of bones - lab bench
19. Blow out marrow
Fill a 10ml syringe with nutrient medium and attach a 22G needle.
Hold tibia at the level of the fibula stump
Insert a needle into the marrow from the proximal end of the tibia and blow out marrow
Some peripheral marrow may remain. Do not crack the bone by pushing the needle too deep.
SECTION: Cleaning of bones - lab bench
20. Rinse
Lift up the tibia, rinse it with spraying medium from needle onto it.
Transfer the tibia to one "Sample" well of the transport plate.
Mark the lid with the code of the sample (please use blinded codes)
SECTION: Cleaning of bones - lab bench
12. Fill one well per tibia in the center of the transport plate (deep round well 96-well plate) with transport medium. Fill the edge or second from edge wells with nutrient medium + antibiotics. 
Cover the plate with lid (but not yet with film). Handle the transport plate with minimizing bacterial contamination.

 

SECTION: Cleaning of bones - lab bench
21. Discard the contents of the 60mm dish, refill with clean nutrient medium and repeat this section for the next bone.
SECTION: Packaging for shipping
23. In a Styrofoam box surround the lidded, sealed transport plate with 4 liquid and 4 solid Cryopak Phase 22 packages. 
SECTION: Consumables and Tools
1. Animal dissection tools:
Serrated/toothed forceps
Scissors
Bone cleaning tools:
Straight tweezers
Curved tweezers
Small scissors
Consumables:
2ml tubes (1 per bone)
15ml and 50ml tubes (2)
10ml syringes (2)
22G needle (1): such as CareTouch hypodermic needle, 22G, 1" #CTHN221
Syringe filter (0.2um) (1)
60ml Petri/culture dish (1)
Lab tape, marker pen
Cryopak phase 22 (8)
Transport plate: 96-well, deep, round-well (~2ml) microplate (1): # Thermo Scientific‱ Nunc‱ 278743
Universal (or matching) plate lid (1): Thermo Scientific‱ Nunc‱ Microplate Lids #250002
Breathable film (2): Excel Scientific BS-25, AeraSeal Sealing Film (Genesee Sci. #12-631)
Common lab tools: pipettes, scale, dissection microscope, tube racks
Reagents: PBS, Custom BGJb nutrient medium, BSA, ITS, penicillin/streptomycin/amphotericin B
SECTION: Cleaning of bones - lab bench
18. Clean off remaining muscle / soft tissues. 
Hold tibia at the distal end, below the fibula stump with tweezers
Pull away or cut off remaining muscle or tendons
Do not peel up the periosteum