May 18, 2026
LVD Lab Transcardial Perfusion Protocol
- 1University of Alabama at Birmingham
Protocol Citation: Laura Volpicelli-Daley, Arielle Manabat, Marissa Menard, Vijay Singh 2026. LVD Lab Transcardial Perfusion Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp77njgzp/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: May 15, 2026
Last Modified: May 18, 2026
Protocol Integer ID: 317285
Keywords: ASAPCRN, lvd lab transcardial perfusion protocol this protocol, lvd lab transcardial perfusion protocol, transcardial perfusions with pb, transcardial perfusion, heparin, na nitroprusside, immunofluorescence, brains for subsequent immunohistochemistry, pb, subsequent immunohistochemistry
Funders Acknowledgements:
ASAP
Grant ID: 020616
Abstract
This protocol describes transcardial perfusions with PBS/Na nitroprusside/Heparin followed by 4% paraformaldehyde. The goal is to fix brains for subsequent immunohistochemistry/immunofluorescence.
Materials
Supplies
- Masterflex Compact Tubing Pump (13-200-001)
- Ismatec Extension Pump Tubing, Puri-Flex (13-200-108)
- Male Stationary Luer Lock and Nut Assembly 1/8" (01-000-429)
- Sterile scalp vein set, 21G (14-840-34)
- Tuberculin syringes (14-841-34)
- Braintree Scientific Tungsten Carbide Iris Scissors, straight, 4.5" (50-195-5882)
- Fine Science Tools Surgical Scissors, Tungsten Carbide, Straight, 14.5 cm (NC9740965)
- Excelta Precision Tweezers with Curved Tips (17-456-112)
- Stir/hot plate
- Thermometer(s): -50°C to +60°C
- pH meter
- Scalp vein set 21G
- Magnetic bar
- Chemical hood
Chemicals
- Isoflurane (Anesthetic) (VetOne NDC 13985-528-60)
- 2-methylbutane (MMX07601)
- Paraformaldehyde powder (Thermoscientific Catalog number A11313.0E)
- Sodium hydroxide (FisherSci #SS267)
- Hydrochloric solution (FisherSci #A144S-500)
- Phosphate buffer saline solution 10X pH 7.4 (FisherSci #50-146-769)
- Sodium pentacyanonitrosylferrate(III) dihydrate (A15656)
- Heparin sodium
Protocol materials
1X PBS
Sodium nitroprussideCatalog #A15656
Heparin sodiumCatalog #25021-400-30
Sodium nitroprusside
30% Sucrose
4% Paraformaldehyde
ddH₂O
Paraformaldehyde (PFA)Thermo ScientificCatalog #AAA113130E
NaOHFisher ScientificCatalog #SS267
HCl
isoflurane
4% PFA
2-methylbutane
10X PBS
Safety warnings
**PFA powder is especially toxic, care should be taken to not breathe it in while measuring and solubilizing, perform as many steps as possible in a fume hood with appropriate PPE.**
Reagent preparation
1X PBS with 0.005 % (v/v) Sodium nitroprusside and 10 U/mL Heparin sodium (1 L )
- 990 mL 1X PBS 1X PBS
- 0.05 g Sodium nitroprussideCatalog #A15656
- 10 mL Heparin sodiumCatalog #25021-400-30
Cryoprotectant Solution: 30 % Sucrose/PBS
30 g 30% Sucrose per 100 mL 1X PBS (final volume)
E.g.,500 mL 30% Sucrose = 150 g sucrose dissolved in 1X PBS then filled to a total of 500 mL solution.
Fixative: 4% Paraformaldehyde /PBS* (See notes to determine volume necessary) (1L)
1. Using an accurate glass beaker or a beaker marked with the correct final volume as measured with an accurate cylinder, add ~80% (~800 mL ) of the desired final volume of ddH₂O to the beaker, add a stir bar and place the beaker on a hot plate (inside fume hood) set to reach around ~60 °C .
a. As the temperature approaches -60 °C (55-60°C) , turn off the hot plate, the temperature does not need to be kept at 60 °C .
b. If the temperature goes above60 °C , wait for it to cool before proceeding.
Safety information
- PFA powder is especially toxic, care should be taken to not breathe it in while measuring and solubilizing, perform as many steps as possible in a fume hood with appropriate PPE.
- Making PFA can be time consuming, preparing all of the reagents the day before is recommended.
Measure Paraformaldehyde (PFA)Thermo ScientificCatalog #AAA113130E out in fume hood so that final concentration will be 4 % (v/v) (e.g., for one liter, 40 g of Paraformaldehyde (PFA)Thermo ScientificCatalog #AAA113130E is added to ddH₂O ).
Add Paraformaldehyde (PFA)Thermo ScientificCatalog #AAA113130E to the hot water with stir bar stirring vigorously and wait for the majority of the Paraformaldehyde (PFA)Thermo ScientificCatalog #AAA113130E to go into solution.
- While waiting for PFA to dissolve, create an ice bath that will accommodate your beaker with ice and water for step 3.5.
Dropwise, add concentrated NaOHFisher ScientificCatalog #SS267 to the solution so that it is no longer cloudy. Small bits floating about are ok.
Move the beaker to your ice bath and monitor temperature until it reaches 25 °C (20-25°C) .
Add 10X PBS to a final concentration of 1X (e.g., for one1 L final volume, 100 mL of 10X PBS is added).
Add ddH₂O to final volume marked on beaker (1000 mL ).
Adjust the pH of the solution to7.4 with NaOHFisher ScientificCatalog #SS267 or HCl .
Filter the solution through 0.2 μm or 0.45 μm filter flask.
Solution can be stored at 4 °C for up to one week or aliquoted to 50 mL tubes and stored in -20 °C for up to 6 months.
Protocol: Perfusion
1d
Anesthetize mouse via isoflurane inhalation described in approved IACUC protocol until mouse no longer responds to tail or toe pinch. Work as quickly as possible after this step.
Transcardially perfuse mouse with cold 1X PBS containing Heparin sodiumCatalog #25021-400-30 (keep on ice) → 25 mL total per mouse at 3 ml/min (Setting “8” on pump).
Note
After you cut the diaphragm, the mouse is no longer alive, but its heart will continue to beat. The sooner you place the needle, the more successful your perfusion will be.
Place the needle through the apex of the heart and keep the needle as straight and to the left (the mouse’s left) as possible to remain in the left ventricle and avoid crossing the heart’s septum.
After the needle is placed, cut the right atrium with scissors, turn on the pump, and manually hold the needle in place until the animal has been perfused with 25 mL of 1X PBS .
Note
- A sign of a good perfusion is when the liver clears of blood and turns to a lighter color.
- If you see liquid drip from the mouse’s nose or mouth, it may indicate the needle was placed too deep in the heart – this doesn’t always result in a poor perfusion, but is something to note if unexpected results occur in downstream experiments.
If 4% PFA perfusion is needed: Perfuse mouse with cold 4% PFA in 1X PBS (keep on ice) → 25 mL total per mouse at 9 ml/min (Setting “8” on pump).
Note
a. Successful circulation of PFA throughout the mouse will often cause the mouse’s tail to move, flick, or stiffen during the perfusion.
b. If perfusing multiple mice in a row, ensure to clear the tubing of excess PFA before beginning the next mouse by running a sufficient volume of 1X PBS solution through the tubing and allowing the PFA to collect in a separate waste container.
For histological analyses: Dissect out desired tissue samples and post-fix in 4% PFA in 1X PBS for 24:00:00 at 4 °C (see Note 11).
1d
Storing 4% PFA-perfused samples
1m
Brain: After post-fixing in 4% PFA in 1X PBS for24:00:00 , rinse tissue with 1X PBS (no sodium nitroprusside/heparin) then place brain tissue in 30% Sucrose in PBS solution until the brain sinks to the bottom of the glass vial (est. 48:00:00 (24 to 48 hours) ). After the brain sinks, snap-freeze the tissue in 2-methylbutane and dry ice (See Note 12) until the brain stops bubbling (est. 00:01:00 (30 to 60 sec) ).
Note
- Store the frozen brains individually wrapped in labeled, aluminum foil, placed in a cardboard box at 80°C.
- For convenience and clear identification, it is recommended to use solvent and temperature resistant printable labels to label each brain wrapped in foil (Tough-Tags Labels on Sheets, 9187-2816).
1m
Notes
1. After placing tissue samples in 4% PFA for post-fixing, immediately place glass vials on ice before storing at 4°C for the 24-hour post-fixing period. Over-fixation of tissues can lead to serious technical difficulties and experimental complications downstream, strict adherence to time and temperature guidelines when tissue is in fixative is vital.
2. Make sure a thermometer is placed in dry ice/2-methylbutane mixture. Ensure the temperature stays between -20 to -30°C for snap-freezing brain tissue.
3. After post-fixing tissue in 4% PFA for 24 hours, rinse with 1X PBS (no sodium nitroprusside or heparin). For cryostat or microtome sectioning, place the 1X PBS-rinsed brains in 30% sucrose until the brain sinks then snap-freeze. For paraffin-embedding, place 1X PBS-rinsed tissue in 70% ethanol until paraffin embedding.
4. Use forceps to carefully place the brain into the 2-methylbutane (kept between -20 to -30°C with dry ice), which should be in a plastic beaker. Temperature of the 2-methylbutane should be monitored the entire time with a thermometer.
5. Assume you will need ~35 mL of 4% PFA per mouse (including post-fix + room for error). Final volume (mL) = # of mice * 35 mL + 100