Jacobs:Protocol Loading Mice
Loading (Days 1-3)
- Turn on Enduratec controllers then turn on computer (pswd: enduratec; Hyperkernel enabled)
- Set up the weigh station
- Assemble anesthesia device
- Change green tubing on O2 tank over to the other side of the room; connect green tubing to isoflurane tank
- Hook up the nose cone to the isoflurane tubing; connect the outlet on the nose cone to a CO2 canister
- Make sure that the outlet on the anesthesia chamber is also connected to a CO2 canister
- Check the isoflurane level in the tank and the weight of the CO2 canister
- Tape the nose cone to the platform, slightly behind the loading cups
- Turn on the O2 (make sure valve going to the chamber is open)
- Set the O2 flow rate at 1.5 L/min (want between 1.5 and 2 L/min for a mouse)
- Set the isoflurane mix at 2.5 (max of 3% isoflurane to O2)
- Open Wintest, using “test” file
- Set Local from “Off” to “High”
- Tare the load by right clicking on the load read-out box and then left clicking on “properties”. Hit Auto and load should be set to 0.
- Confirm that program is on “displacement” feedback control
- Click on position tab and set the loading cup to -2.5
- Anesthetize the first mouse in the chamber
- Once the mouse is fully anesthetized, switch the isoflurane from the chamber to the nose cone. Then place the mouse atop the platform, sticking its nose inside the nose cone.
- Insert the right ulna into the loading cups
- Hold the paw with your right hand; use your left thumb to pull away fur under the elbow
- Once elbow is visibly poking out, guide it into the lower loading cup
- Gently push the wrist into the upper loading cup, adjusting the position as necessary
- Make sure that the hand is bent downwards towards the platform
- Lower the loading cup using the position tab until the pre-load is between -0.1 and -0.2 (typically this corresponds to a position between -2.5 and -3.5)
- Change Feedback from “displacement” control to “load” control
- Check Waveform
- Level 1: -0.1 (preload); Level 2: -3.0 (or desired load level)
- Frequency: 2 Hz; Cycles: 120
- Done Action: Stop Level
- Run: ZeroStart
- When loading has finished:
- Switch the isoflurane back from the nose cone to the chamber
- Switch feedback from “load” control to “displacement” control
- Using the “position” tab, move the upper loading cup back to it’s original position (typically between -2.0 and -2.5)
- Remove the mouse ulna from the device and place mouse inside the weighing station for recovery
- Record the mouse weight and time to recovery
- Repeat steps 5 through 13 on all mice
- Anesthetize mouse
- (Feedback control to “displacement”; change position to -2.5)
- Switch isoflurane from chamber to nose cone
- Insert mouse ulna and apply preload of -0.1 N
- Feedback control to “load”
- Check waveform (make sure done action is at “stop level”)
- Run: ZeroStart
- Feedback control to “displacement”; change position back to -2.5
- Switch isoflurane from nose cone to chamber
- Weigh mouse and record recovery
- When finished with all mice:
- Turn off O2 tank and isoflurane chamber; release excess O2
- Clean all chambers and platform with disinfectant (not alcohol!)
- Turn off computer and then 2 controllers
**Remember to re-tare load every now and then!
CO2 canisters: From Laura at the VMU; change when they’ve accumulated 50 g in mass; activated carbon used to absorb the isoflurane and CO2 that the mouse exhales Isoflurane: Stored in flammables cabinet in Derek’s lab O2 tank: Call Derek when you switch to the new tank (or when the dial reaches the “refill” red zone)
Injecting mice with Calcein (Day 5)
- Want to inject 10 mg/kg (calcein/mass of mouse); Calcein (Sigma 0875-5G; IR, G-11)
- Make working solution: Mix 25 mg (0.025 g) of calcein in 10 mL PBS and vortex; can store for 2 wks at 4˚C; light sensitive, so wrap in foil
- Calculate 4 x (mass of mouse) = (vol in μL to inject into mouse)
Take syringes, calcein, and needles to the VMU
- Use ≥27 gage needle for sub-cutaneous mouse injection. We typically use 27½ G needle (B-D 305109)
- Use a 1 mL syringe (Tyco/Kendall 8881501400; Monoject Tuberculin syringes w/o needle)
- Prepare for injection by pulling a volume of calcein into the syringe, flip over and tap bubble to top of syringe; attach needle to syringe and squirt out some solution to remove any air bubbles; note the starting volume on the syringe
- Scruff a mouse well with your right hand; fold tail between the ring finger and the pinky finger
- With left hand, gently push needle into the skin between your finger tips; inject approximately 0.1 to 0.15 mL, depending on the mass of the mouse
- Make sure mouse recovers well and then dispose of needle in sharps container
Injecting mice with Alizarin Red (Day 9)
- Want to inject 50 mg/kg; Alizarin Red S, Certified (Sigma A5533-25G; IR, G-5, Cas 130 22 3)
- Make working solution: Mix 125 mg (0.125 g) to 10 mL PBS and vortex; can store for 2 wks at 4˚C; light sensitive, so wrap in foil
- Follow same procedure as for sub-cutaneous calcein injection
Sacrifice (Day 15)
- Sac mouse using CO2 and cervical dislocation
- Start with right ulna; cut incision at right armpit and pull off fur & skin from right ulna
- Cut arm at the midshaft of the humerus
- Place elbow on table and bend wrist such that the lateral tendons are pulled across the wrist; carefully trim tendons at the wrist.
- Hold paw and carefully pull the tendons and muscle down the ulna to separate away from the bone; cut off excess muscle and tendons.
- Use a piece of gauze to pull away any additional muscle and other tissue attached to the ulna; can use scissors to carefully cut off soft tissue.
- Trim muscle and fat off of the humerus.
- Holding the ulna with your right hand, gently wiggle the humerus using tweezers in your left hand until the humerus is free from the ulna socket. Take special care not to break the ulna.
- After the humerus has been removed, clean any remaining tissue from the ulna.
- For the right ulna, measure the total length of the bone and then make a mark with a pencil at the midpoint of the bone.
- Repeat the procedure for the left ulna. Record the total length of the bone, but do not make a mark at the midpoint.
- Remove the radius on the left ulna: gently wedge a scalpel tip between the radius and ulna until they have separated. Take special care not to break the ulna
- Bind the left and right ulnae together using bare copper wire (32 awg)
- Wrap wire around elbow sockets (first) and wrists (second) – only 2 wraps per location!
- Twist wire back on itself and cut off free ends
- Cut off the distal and proximal tips of the ulnae (at the growth plate) to allow for better penetration of the fixative
- Fix bones in 70% EtOH (approx 20x more fixative than bone)
- Store at RT in 70% EtOH
- History: CMBL – JBL (from JT and ST), last updated 4/08
or instead, discuss this protocol.