Nov 13, 2025

Public workspaceVessel Painting Perfusion Protocol for Mice (Adults and Pups)

DOI
https://dx.doi.org/10.17504/protocols.io.j8nlkypo5g5r/v1
  • Gustavo A. Gomez1,
  • Amandine Jullienne1,
  • Terese Garcia1,
  • JiaMin Yan1,
  • Paul R. Territo2,
  • Andre Obenaus1
  • 1Division of Biomedical Sciences, School of Medicine, University of California, Riverside;
  • 2Stark Neuroscience Research Institute, Division of Clinical Pharmacology, School of Medicine, Indiana University
  • TBI ADRD UCR/IU
gustavo.gomez
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DOI: https://dx.doi.org/10.17504/protocols.io.j8nlkypo5g5r/v1
Protocol Citation: Gustavo A. Gomez, Amandine Jullienne, Terese Garcia, JiaMin Yan, Paul R. Territo, Andre Obenaus 2025. Vessel Painting Perfusion Protocol for Mice (Adults and Pups). protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkypo5g5r/v1
Manuscript citation:
Salehi et al 2018. https://doi.org/10.1007/s12975-018-0632-0
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: November 06, 2025
Last Modified: November 13, 2025
Protocol Integer ID: 231697
Keywords: Mouse, Vessel Painting, Carbocyanine dyes, Intracardiac Perfusion, vessel painting perfusion protocol for mice, vessel painting perfusion protocol, perfusion fixation for mice, process for vessel painting, vessel painting, perfusion fixation, mice, separate sop, standard operating procedure, sop, process
Funders Acknowledgements:
NIH NINDS
Grant ID: 1RF1NS138032-01
NIH NINDS
Grant ID: 5R01NS135556-03
NIH NINDS
Grant ID: 7R01NS119605-04
Disclaimer
The authors have nothing to disclose
Abstract
This standard operating procedure (SOP) document describes the process for vessel painting followed by perfusion fixation for mice (adults and pups). Data analysis routes are not included here and are identified in a separate SOP.
Attachments
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Appendix 1.pdf
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Appendix 2.pdf
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Appendix 3.pdf
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Appendix 4.pdf
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Appendix 5.pdf
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Image Attribution
Graphs in Appendix 3 were produced by Biotium
Materials
Equipment needed:
• Peristaltic perfusion pump (MSE Supplies, # LS0332-4)
• Eppendorf Tube warmer (Thermo Scientific, #88870001)
• Perfusion tray and instruments (Scissors, forceps, clamp applying forceps with lock F.S.T. #00071-14, dissection pins, curved hemostat forceps)
• 25-gauge butterfly needle (BD Vacutainer, #367285). Note: blunt the tip of the needle with a Dremel rotary tool before use
• 0.5 mL, Insulin Syringe with needle, 28 Gauge X ½ Inch (BD, #329461)
• Induction chamber, isoflurane vaporizer, flow meter, air pump
• Weighing Scale
• Two 1-Liter Glass Bottles for perfusion solutions [(A) 1X PBS and (B) 4%PFA]
• Plastic tubing is used to drive perfusion solutions into the peristaltic pump and away from the pump to perfuse the animal as shown in Salehi et al. 2018, with a modification: A 3-way stopcock valve is fitted between the plastic tubing connected to either 1X PBS or 4% PFA. (see pic)
• Vessel Painting (VP) perfusion data sheet to note weight, perfusion quality, any comments, etc. (Download Appen
• 20 mL HDPE Scintillation Vials (Fisherbrand #03-337-23C) for head collections
• 1.5 mL microcentrifuge tube (Fisherbrand # 05-408-129): used to mix diluent and dye, other types of tube might lead to formation of aggregates
• Isoflurane vaporizer (Kent Scientific, #VETFLO-1210S)
• 14 mm rodent nose cone (Avantor # 89012-602)

Reagents Needed:
• Isoflurane anesthetic inhalant USP (Covetrus #029405)
• 1X PBS (pH 7.4), Dissolve Calcium- and Magnesium-free PBS tablets (MP Biomedicals #092810307) in dH2O water (1 tablet per 100mL), then measure pH. Use within 2 weeks.
• 10X PBS (pH not adjusted): also made with PBS tablets (1 tablet in 10 mL, MP Biomedicals #092810307).
• PFA 4% (pH 7.4, made from 32% solution (EMS #15714-S) in PBS 1x, pH 7.4).
ο Example for 1000 mL 4% PFA: Dissolve 10 PBS tablets in 875 mL ddH2O. Stir the solution with a stirring rod on a stir plate, then add 125 mL of 32% PFA while stirring. Finally, adjust the pH of the solution to 7.4. Store at 4°C when not in use, but warm to room temperature before beginning mouse perfusion/fixation. Use within 2 weeks.
ο PFA is Hazardous! Operate only in a fume hood
• SNP (sodium nitroprusside, dihydrate, Sigma Aldrich, #71778) solution (0.735 mg/mL in 1X PBS). Example: Dissolve 1.3 mg SNP in 1.768 mL 1X PBS. Must be used within 1 week, and stored at 4°C when not in use.
• Heparin sodium injection 1000 USP units/mL (McKesson, Cat #916433)
• Dye Stock solution (DiI, DiD or DiO) dissolved to a final concentration of 2.99mg/mL
ο 100 mg DiI (LifeTechnologies #D282) + 33.4 mL Ethanol 200 proof (Fisher #BP2818)
ο 50 mg DiO (Biotium, Cat #60012) +16.7 mL Ethanol 200 proof
ο 100 mg DiD (Invitrogen #D7757) + 33.4 mL Ethanol 200 proof
ο 25 mg Neuro-DiO (Biotium, Cat #60015) + 8.35 mL Ethanol 200 proof
• Diluent solution (2% dextrose (Fisher D14) + 1 mL of 10X PBS + 49 mL ddH2O). Keep at 4°C for no longer than one week
• Sodium Azide (Sigma-Aldrich # S2002-100G) for long-term storage of vessel painted heads. Prepared as a 0.02% solution in 1X PBS
ο Sodium Azide: Toxic-Hazardous! Work under fume hood
• Evans Blue (Fisher Scientific #AC195550050)
• Saline (Hudson RCI Addipak 0.9% Saline solution #200-39) for dissolving Evans Blue (if used). Note: Evans Blue is dissolved to 2% final concentration (w/v) in saline and filtered with 0.22 μm Cellulose Acetate membrane filter (Corning #430767) before IP injection
Troubleshooting
Safety warnings
• PFA is Hazardous! Operate only in a fume hood
• Sodium Azide: Toxic-Hazardous! Work under fume hood
• Do not collect blood if Evans Blue was injected.
Ethics statement
This protocol follows institutional regulations consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the ARRIVE guidelines for reporting animal research. Experimental procedures involving animals comply with protocol No. 30608, approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, Riverside, School of Medicine, and protocol No. 25046 approved by IACUC at Indiana University.
Before start
Scope
The scope of this SOP includes:
  • Solutions required to successfully complete vessel painting, perfusion, and fixation
  • Equipment setup and solution recipes
  • The euthanasia procedure
  • The surgical procedure
  • Post-fixation methods
  • Attention items are written in Italicized Bold

Responsibilities
  • SOP Author: responsible for production of the SOP and its described procedures
  • SOP Owner: responsible for reviewing and approving changes to the SOP
  • Staff: responsible for adhering to SOP guidelines
  • NO CHANGES to this SOP are authorized without the explicit approval of the SOP Owner (See Appendix 1)

Preparation before starting (These steps are re-iterated in step 3 of the Procedure):

Evans Blue Injection
• If Evans Blue is injected to assess degree of blood-brain barrier disruption resulting from the Closed Head Injury, a 2% solution is injected by IP 2 hours before Vessel Painting/Perfusion at 3 μL/g of animal weight. (See VP perfusion data sheet- Appendix 4, 5).

Perfusion set up (Perform in the safety cabinet/fume hood)

• Fill a 1 Liter bottle with at least 25 mL of 1X PBS (pH 7.4) solution per mouse.
• Prepare the second 1L bottle, or flask with at least 25 mL (per mouse) of 4% PFA.
• Submerge plastic tubing connected to the 3-way stopcock valve into either 1X PBS or 4% PFA.
• Run solutions through the line eliminating air bubbles, and adjust the flow rate to 5 mL/min for pups or 8 mL/min for adults. First, run the 4% PFA solution through the tubing until it is dispensed at the butterfly needle. With the pump running, turn the stopcock to run PBS through the tubing until enough solution is dispensed to flush out any remaining PFA from the line in preparation to perfuse the first animal (this takes about 1 minute).

Preparation of the dye for injection (This is done on a benchtop outside of the safety cabinet)

• Prepare dye by warming the diluent to 37°C on the Eppendorf tube warmer. Warm diluent individually for each mouse: 5-7minutes minimum in a graduated 1.5 mL microcentrifuge tube. Mix the diluent and dye stock solution (see Table 1 for volumes) right before injection by vortexing for 10 seconds at maximum power. Then load the solution into a 0.5mL insulin syringe. Make sure to remove ALL air bubbles in the syringe before injecting the dye into the left ventricle of the heart. The same syringe can be used for up to 10 animals at a time.
Procedures
Flow-Chart: an outline of the procedures performed in this SOP




Equipment needed:
• Peristaltic perfusion pump (MSE Supplies, # LS0332-4)
• Eppendorf Tube warmer (Thermo Scientific, #88870001)
• Perfusion tray and instruments (Scissors, forceps, clamp applying forceps with lock F.S.T. #00071-14, dissection pins, curved hemostat forceps)
• 25-gauge butterfly needle (BD Vacutainer, #367285). Note: blunt the tip of the needle with a Dremel rotary tool before use
• 0.5 mL, Insulin Syringe with needle, 28 Gauge X ½ Inch (BD, #329461)
• Induction chamber, isoflurane vaporizer, flow meter, air pump
• Weighing Scale
• Two 1-Liter Glass Bottles for perfusion solutions [(A) 1X PBS and (B) 4%PFA]
• Plastic tubing is used to drive perfusion solutions into the peristaltic pump and away from the pump to perfuse the animal as shown in Salehi et al. 2018, with a modification: A 3-way stopcock valve is fitted between the plastic tubing connected to either 1X PBS or 4% PFA. (see pic)


• Vessel Painting (VP) perfusion data sheet to note weight, perfusion quality, any comments, etc. (Download Appen
• 20 mL HDPE Scintillation Vials (Fisherbrand #03-337-23C) for head collections
• 1.5 mL microcentrifuge tube (Fisherbrand # 05-408-129): used to mix diluent and dye, other types of tube might lead to formation of aggregates
• Isoflurane vaporizer (Kent Scientific, #VETFLO-1210S)
• 14 mm rodent nose cone (Avantor # 89012-602)
Reagents Needed
• Isoflurane anesthetic inhalant USP (Covetrus #029405)
• 1X PBS (pH 7.4), Dissolve Calcium- and Magnesium-free PBS tablets (MP Biomedicals #092810307) in dH2O water (1 tablet per 100mL), then measure pH. Use within 2 weeks.
• 10X PBS (pH not adjusted): also made with PBS tablets (1 tablet in 10 mL, MP Biomedicals #092810307).
• PFA 4% (pH 7.4, made from 32% solution (EMS #15714-S) in PBS 1x, pH 7.4).
ο Example for 1000 mL 4% PFA: Dissolve 10 PBS tablets in 875 mL ddH2O. Stir the solution with a stirring rod on a stir plate, then add 125 mL of 32% PFA while stirring. Finally, adjust the pH of the solution to 7.4. Store at 4°C when not in use, but warm to room temperature before beginning mouse perfusion/fixation. Use within 2 weeks.
ο PFA is Hazardous! Operate only in a fume hood
• SNP (sodium nitroprusside, dihydrate, Sigma Aldrich, #71778) solution (0.735 mg/mL in 1X PBS). Example: Dissolve 1.3 mg SNP in 1.768 mL 1X PBS. Must be used within 1 week, and stored at 4°C when not in use.
• Heparin sodium injection 1000 USP units/mL (McKesson, Cat #916433)
• Dye Stock solution (DiI, DiD or DiO) dissolved to a final concentration of 2.99mg/mL
ο 100 mg DiI (LifeTechnologies #D282) + 33.4 mL Ethanol 200 proof (Fisher #BP2818)
ο 50 mg DiO (Biotium, Cat #60012) +16.7 mL Ethanol 200 proof
ο 100 mg DiD (Invitrogen #D7757) + 33.4 mL Ethanol 200 proof
ο 25 mg Neuro-DiO (Biotium, Cat #60015) + 8.35 mL Ethanol 200 proof
• Diluent solution (2% dextrose (Fisher D14) + 1 mL of 10X PBS + 49 mL ddH2O). Keep at 4°C for no longer than one week
• Sodium Azide (Sigma-Aldrich # S2002-100G) for long-term storage of vessel painted heads. Prepared as a 0.02% solution in 1X PBS
ο Sodium Azide: Toxic-Hazardous! Work under fume hood
• Evans Blue (Fisher Scientific #AC195550050)
• Saline (Hudson RCI Addipak 0.9% Saline solution #200-39) for dissolving Evans Blue (if used). Note: Evans Blue is dissolved to 2% final concentration (w/v) in saline and filtered with 0.22 μm Cellulose Acetate membrane filter (Corning #430767) before IP injections
Preparation
Evans Blue Injection
• If Evans Blue is injected to assess degree of blood-brain barrier disruption resulting from the Closed Head Injury, a 2% solution is injected IP, 2 hours before Vessel Painting/Perfusion at 3 μL/g of animal weight. (See VP perfusion data sheet).

Perfusion set up (Perform in the safety cabinet/fume hood)
• Fill a 1 Liter bottle with at least 25 mL of 1X PBS (pH 7.4) solution per mouse.
• Prepare the second 1L bottle, or flask with at least 25 mL (per mouse) of 4% PFA.
• Submerge plastic tubing connected to the 3-way stopcock valve into either 1X PBS or 4% PFA.
• Run solutions through the line eliminating air bubbles, and adjust the flow rate to 5 mL/min for pups or 8 mL/min for adults. First, run the 4% PFA solution through the tubing until it is dispensed at the butterfly needle. With the pump running, turn the stopcock to run PBS through the tubing until enough solution is dispensed to flush out any remaining PFA from the line in preparation to perfuse the first animal (this takes about 1 minute).

Preparation of the dye for injection (This is done on a benchtop outside of the safety cabinet)
• Prepare dye by warming the diluent to 37°C on the Eppendorf tube warmer. Warm diluent individually for each mouse: 5-7minutes minimum in a graduated 1.5 mL microcentrifuge tube. Mix the diluent and dye stock solution (see Table 1 for volumes) right before injection by vortexing for 10 seconds at maximum power. Then load the solution into a 0.5mL insulin syringe. Make sure to remove ALL air bubbles in the syringe before injecting the dye into the left ventricle of the heart. The same syringe can be used for up to 10 animals at a time.

Table 1. Dye and Diluent- Volumes to mix


Anesthesia Procedure:
• Administer SNP + heparin (See Table 2 for volumes) by intraperitoneal (IP) injection with a 0.5 mL syringe before placing the mouse into the isoflurane induction chamber. Use a separate syringe to inject each mouse.
• Anesthetize the animal with isoflurane in air for 5 min (3% Isoflurane, air flow: 1.5 L/min).
• Weigh the animal and record its weight on the VP perfusion data sheet.
Table 2. SNP and Heparin- Volumes to mix


Surgical Procedure
• Once the mouse has reached deep anesthesia and does not respond to a toe pinch, place it on the tray and stabilize it by pinning the mouse with dissection pins at all four limbs.
ο The head of the mouse should be placed in a nose cone throughout the surgical procedure to continue isoflurane anesthesia.
• Make an incision below the sternum. Expose the thoracic cavity by cutting below the rib cage and moving to a lateral position. Puncture and cut the diaphragm then cut through ribs on both sides. Note: Cutting the diaphragm will result in cessation of oxygen intake, the next steps need to be performed quickly!
• Use a curved hemostat to hold the rib cage up and expose the heart, taking care not to damage the lungs.
ο Do not collect blood if Evans Blue was injected.
• If collecting blood, insert a 25-gauge needle attached to a 1mL syringe into the left ventricle, then slowly collect about 200-300μL of blood (see plasma collection SOP).
• Using the same initial puncture hole (if blood was collected), insert the 0.5mL syringe pre-filled with dye into the left ventricle.
ο It is crucial to use the same puncture hole for adequate vessel painting/perfusion
• Inject dye solution (DiI, DiD, or DiO) over 10 seconds, then allow the dye to circulate through the mouse for 15 seconds.
• The heart should still be beating during dye injection. If the heart is not beating, use forceps to gently mechanically compress the heart once the needle is removed from the heart. Note on the VP perfusion data sheet if manual compression was required.
• Insert the 25-gauge butterfly needle from the pump line into the puncture entry in the left ventricle of the heart.
• Secure the inserted butterfly needle position using the forceps with the clamp (F.S.T. #00071-14), then cut a hole in the right atrium for liquids to drain.
• Run the pump according to age-specific parameters as listed below:
ο Use a timer to keep track of perfusion and fixation times
ο Immediately switch the stopcock valve at the intake line when switching from PBS to PFA
∗ PUPS: pump flow rate setting = 5 mL/min
∗ For 10 mL 1X PBS; run the pump for 2 minutes
∗ For 16 mL 4% PFA: run the pump for 3 minutes: 20 seconds
∗ ADULTS: pump flow rate setting = 8 mL/min
∗ For 10 mL 1X PBS: run the pump for 1 minute: 15 seconds
∗ For 20 mL 4% PFA: run the pump for 2 minutes: 30 seconds
• 30 seconds longer for both age groups if the mouse is not stiff after the volumes listed.
• Turn off the pump, remove the butterfly needle from the heart, and behead the animal at the mid-cervical vertebrae (above the shoulders).
• Place the decapitated head into a 20 mL scintillation tube filled with 4% PFA.
• Rinse the tubing and butterfly needle with 1X PBS by switching the valve at the intake line to the 1X PBS solution and run the pump for 2 min to clear PFA from the system in preparation for the next mouse.
Post-fixation Procedure
• Submerge head/brain in 4% PFA solution for 24 hrs at 4°C in the 20mL Scintillation Vial. Replace the PFA solution with 1X PBS and store at 4°C for 24 hours. Repeat this PBS wash two more times, for a total of 3 washes with 1X PBS. For long-term storage, transfer to 0.02% sodium azide (in 1X PBS), and store at 4°C.
• Document whether 1) heads have been fixed in 4% PFA for one day, 2) washed in 1X PBS three days thereafter, and 3) whether they were transferred to 0.02% sodium azide/PBS. This can be done by making marks directly on the lids of the vials, research notebooks, and Excel spreadsheets.
References
Salehi et al 2018. https://doi.org/10.1007/s12975-018-0632-0
Appendices
Appendix 1: SOP Record Log
Appendix 2: Examples of vessel painted mouse brains with different dyes
Appendix 3: Excitation-Emission Spectra for different Lipophilic Carbocyanine Dyes
Appendix 4: VP Perfusion Data Sheet for Adults
Appendix 5: VP Perfusion Data Sheet for Pups
Protocol references
Salehi et al 2018. https://doi.org/10.1007/s12975-018-0632-0