Jan 17, 2025
Version 1
Mouse perfusion with JF dye at surgical plane of anesthesia V.1
- 1HHMI Janelia Research Campus
Protocol Citation: Boaz Mohar, Monique Copeland 2025. Mouse perfusion with JF dye at surgical plane of anesthesia. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g71ej3gwz/v1
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: October 01, 2024
Last Modified: January 17, 2025
Protocol Integer ID: 108731
Keywords: mouse, perfusion, HaloTag
Funders Acknowledgements:
HHMI
Abstract
This protocol is aimed at improving the reliability delivery of small molecules (JF dyes and others) to the brain during perfusion.
Materials
MATERIALS
Disposable gloves, nitrile
Surgical ScissorsFine Science ToolsCatalog #14090-09
Standard Pattern ForcepsFine Science ToolsCatalog #11001-12
ForcepsFine Science ToolsCatalog #11223-20
IsofluranePenn Veterinary Supply, Inc.Catalog #CED1360
STEP MATERIALS
Dimethyl sulfoxide (DMSO)Sigma AldrichCatalog #D2650
Falcon Tube (50 mL)Fischer Scientific
Isoflurane 1-3%Patterson VeterinaryCatalog #07-893-1389
PBSFisher ScientificCatalog #BP24384
Equipment
United Scientific™ Aluminum Dissecting Pan with Black Wax
NAME
Dissection tray
TYPE
United Scientific
BRAND
S111022
SKU
LINK
Equipment
Watson-Marlow 120S Variable-Speed Single-Channel Pump System
NAME
Peristaltic pump
TYPE
Watson Marlow
BRAND
EW-50130-00
SKU
LINK
Equipment
Surgical Scissors
NAME
Fine Science tools
BRAND
14090-09
SKU
LINK
Equipment
Forceps
NAME
Fine Science tools
BRAND
11223-20
SKU
LINK
Equipment
Surflo winged infusion set 21G 3/4
NAME
Terumo
BRAND
TER 3SV-21BLK
SKU
LINK
Prepare dye and fixative
Prepare dye and fixative
1h
1h
Add 100 µL of DMSO to lyophilized dye (100nmol) and pipette up and down ~20 times to get 1mM concentration stock.
Make sure to use anhydrous DMSO stored under desiccation after opening.
Mix further using a vortex machine to ensure the dye is evenly distributed in the DMSO
Make sure there is no concentrated dye pellet after vortex in the bottom of the tube.
Add up to 25 µL 1 mM JF dye stock to 50 mL 4% paraformaldehyde in .1M PB and mix by inverting 5 times. Set aside for later use. Make sure no dye is seen at the bottom of the tube as you start the perfusion.
The amount of dye depends on the amount of HaloTag protein in you mouse and its distribution. 25ul is an upper limit in our hands, less can work.
Set-up down draft table and anesthesia system for perfusion
Set-up down draft table and anesthesia system for perfusion
Set up down draft table with induction box, dissection tray, perfusion pump, and tools.
Induction chamber
Replace paper towel between mice.
**Optional: Place a quarter inch piece of vinyl mat in the dissection tray to elevate the moue for better placement of the nose cone.
Set up the anesthesia system.
Check the oxygen and isoflurane lines to the system and verifying the flow to the nose cone is closed and the flow to the induction box is open.
Check the level of isoflurane and fill if needed. Open the oxygen flow from the compressed tank supply.
Secure the nose cone to the upper edge of the dissection tray using tape. Make sure the nose cone will not shift during the procedure so the mouse's nose and mouth remain in the cone.
Clean the nose cone between mice as fixation solution could come out the nose of the mouse and should not contact the nose of the next mouse.
Perfusion and dissection
Perfusion and dissection
4m
4m
Prime the perfusion pump by attaching the needle to pump tubing and flush with ~10 mL of 1x PBS to clear tubing of previous solutions. Transfer tubing to 50ml tube with 4% paraformaldehyde.
**For our purposes the tubing length is 20 inches and holds 10mL of fluid. You may need to flush additional 1x PBS prior to transferring tubing to PFA depending on tubing length and diameter.
**Residual dye may remain in the tubing after flushing with 1x PBS. It is recommended to keep separate tubing for each dye. Label tubing with specific dye and store in a dry place between perfusions.
**When transitioning between perfusion solutions make sure to stop the pump prior to moving to tubing to prevent bubbles from forming in the lines. Bubbles perfused though the mouse can cause deformations in the ventricles.
Retrieve animal per vivarium protocol.
Start the oxygen flow to the induction box at 1 L/min while the isoflurane remains at 0%
Perfusion and dissection
Perfusion and dissection
4m
4m
Quickly place the mouse in the induction box and secure the lid.
Set the isoflurane to 3% and monitor the mouse.
Anesthetize the mouse according to your IACUC protocol.
For our purposes we anesthetize the mouse for 3-4 minutes in the induction box. The mouse should lay still and its breathing pattern will reduce to ~1 breath/second.
4m
Lower the concentration of the isoflurane to 2% and the oxygen flow to 0.8 L/min
Open the oxygen/isoflurane flow to the nose cone and close the flow to the induction box.
Quickly remove the mouse from the induction box and place on the vinyl mat in the dissection tray. Placing the mouse's nose and mouth in the nose cone. Hold the mouse in place with your hand for around00:01:00
1m
Check for sufficient level of anesthesia.
1. Check for lose of muscle tone by extending the hind limb and note retraction.
2. Check for pedal reflex by gently pinching the mouse's foot.
If the animal reacts, continue hold the mouse in place. If no reaction pin the mouse with one pin in
each arm and others as needed, so the nose and mouth remain in the nose cone.
Check the pedal reflex again by pinching the foot. If the mouse reacts wait and check again in 30
seconds. Continue to check until the mouse does not react.
Carefully use scissors to open the chest cavity by lifting the base of the sternum and cutting parallel to either side of the sternum to the neck, then cut the diaphragm, making sure to avoid puncturing the heart, and expose the heart.
The blood will appear bright red as it is oxygenated, and the heart will beat rapidly.
Gently hold the heart by the left ventricle and insert tip of the butterfly needle into the bottom tip of the right ventricle and cut the left atrium to exsanguinate the mouse.
Optional use a 18G needle to secure the butterfly needle in place or a hemostat clamp.
Perfusion and dissection
Perfusion and dissection
4m
4m
Start perfusion pump and flush the 10mL of 1xPBS primed in the tuning followed by 50 mL 4% PFA with up to 25 µL of JF dye at a rate of 7.5ml/min.
The amount of dye depends on the amount of HaloTag protein in you mouse and its distribution. 25ul is an upper limit in our hands, less can work.
Animal will have muscle contractions when the fixative circulates through the bloodstream. If no reaction occurs check position of the needle in the heart. During the muscle contractions the intestines and liver may extend outside of the abdominal cavity causing the needle to shift.
10m
"Stop" perfusion pump when all fixative is flushed through the animal.
Unpin mouse from dissection tray and decapitate behind ears.
(Place carcass in small biohazard bag for disposal.)
Prepare the post-fix of 50 mL 4% PFA with 25 µL JF dye tube labeled with the animal ID, date, and 4% PFA
The amount of dye depends on the amount of HaloTag protein in you mouse and its distribution. 25ul is an upper limit in our hands, less can work.
Remove the fur from the head and cut small slits into the skull by placing scissor tip on eithier side of the opening where the spinal cord connect to the brain.
Use forceps to peel off skull cap and gently scope out brain.
Perfusion and dissection
Perfusion and dissection
4m
4m
Double check the animal ear tag with the cage card to confirm animal ID on the tube is correct then place the brain in the post-fix for 08:00:00 or Overnight 45 rpm, 4°C
8h
Perfusion and dissection
Perfusion and dissection
4m
4m
Rinse 4 times 00:15:00 00:15:00 Overnight Overnight 45 rpm, 4°C in 50ml 1x PBS.
Extended rinses are needed to make sure excess dye from the perfusion and postfix are cleared.
30m