Intracardiac perfusion fixation of experimental mice: preparation of tissue specimens for anatomical studies
Browse experimental protocolsAdult mice are anesthetized by intraperitoneal injection of ketamine (150 mg/kg) with xylazine (15 mg/kg); the chest is opened and a needle connected to a peristaltic pump is placed in the left ventricle, and a prewash solution followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) is perfused. A step in the flow labels the arterial and capillary systems in vivo with DyLight 649-conjugated tomato lectin injected into the left ventricle. Dissected tissues are post-fixed at 4 °C for 18–24 h, washed in 0.1 M PBS (pH 7.2) and stored at 4 °C in PBS containing 0.1% sodium azide until used for immunohistochemistry or other microscopic analysis, yielding well-preserved tissue for anatomical studies. Institutional animal ethics approval must be obtained before performing this procedure.
Scope and method
Sample：Adult mouse tissues (brain, spinal cord, ganglia, and internal organs)
- C57 mouse MCAO suture model
- Age
- adult
- Tissue
- brain
- 5xFAD transgenic model
- Age
- adult
- Tissue
- brain
- 3xTg-AD transgenic model
- Age
- adult
- Tissue
- brain
Bill of materials (BOM)
| Step | Material | Role | Amount | Required / optional | Substitution conditions | SKU / specification | Literature product |
|---|---|---|---|---|---|---|---|
| Thoracotomy and left ventricular cannulation | Perfusion needle, tubing and T-connector assembly | Used for left ventricular cannulation, connecting prewash and fixative solutions to the perfusion tubing through a T-connector. Assembly comprising a perfusion needle, connecting tubing and a T-connector; the source does not report needle gauge, tubing diameter or materials. | Not reported in the source | Required | Not reported in source | Available for inquiry | Not reported in source |
| Labeling of the arterial and capillary systems | 31G injection needle for vascular lectin labeling | Used to inject lectin into the left ventricle in the vascular-labeling branch. Needle gauge 31G; used only when vascular labeling is selected and not required for every perfusion. | Not reported in the source | Optional | Not reported in source | Available for inquiry | Not reported in source |
| Induction of anesthesia | Ketamine (HCl) | Anesthesia induction Anesthetic for experimental animal injection, administered in ketamine-based dosage; source record labeled Lyppard #KETAI1. | Ketamine 150 mg/kg, intraperitoneal injection | Required | Not reported in source | Available for inquiry | Not reported in source |
| Prewash perfusion | Perfusion pre-wash solution (sodium chloride-sodium nitrite-heparin mixture) | Flush blood before perfusion Add sodium nitrite and heparin (5000 IU/mL) to 0.9% sodium chloride immediately before use; perfuse with a peristaltic pump at approximately 18 mL/min. | Adult mice typically have about 18–20 mL; perfuse until the effluent is clear and the liver and limbs are pale. | Required | Not reported in source | Available for inquiry | Not reported in source |
| Fixative perfusion | 4% paraformaldehyde/0.1 M phosphate buffer fixative (pH 7.4) | Perfusion fixation 4% paraformaldehyde is prepared in 0.1 M phosphate buffer (pH 7.4); use within no more than 48 hours after preparation, store at 4°C, and bring to room temperature on the day of perfusion. | 90 mL per adult mouse | Required | Not reported in source | Available for inquiry | Not reported in source |
| PBS washing | 0.1 M phosphate buffer (pH 7.2) rinse solution | Fixed tissue rinsing PBS concentration 0.1 M, pH 7.2 (distinct from the pH 7.4 formulation of existing entries on this site). | Each immersion rinse lasts 30 minutes, for a total of 3 times. | Required | Not reported in source | Available for inquiry | Not reported in source |
| Induction of anesthesia | Xylazine (HCl) | Anesthesia induction (in combination with ketamine) Experimental animal injectable drug, used in combination with ketamine; source record labeled Lyppard #L10605. | Xylazine 15 mg/kg, intraperitoneal injection | Required | Not reported in source | Available for inquiry | Not reported in source |
| Tissue collection and postfixation | 4% paraformaldehyde/0.1 M phosphate buffer fixative (pH 7.4) | Fix after tissue collection Same formulation as the perfusion fixative (4% paraformaldehyde/0.1 M PB, pH 7.4). | Each tissue sample was immersed in fixative and fixed at 4°C for 18–24 hours. | Required | Not reported in source | Available for inquiry | Not reported in source |
| Labeling of the arterial and capillary systems | DyLight 649-labeled Tomato Lectin (Lycopersicon esculentum) | In vivo labeling of the arterial and capillary systems Conjugate labeled with DyLight 649 fluorescence, source record annotated as Thermo Fisher Scientific #L32472; perform this step immediately after thoracotomy only when vascular labeling is required. | Inject 100 µL of 1 mg/mL working solution into the left ventricle within 30 seconds, and circulate for 1 minute. | Optional | Not reported in source | Available for inquiry | Not reported in source |
| Storage | PBS storage solution containing 0.1% sodium azide | Tissue preservation before testing Contains 0.1% sodium azide; sodium azide inhibits horseradish peroxidase (HRP) and is incompatible with HRP-based detection systems. | Tissue was immersed in preservation solution and stored at 4°C | Required | Not reported in source | Available for inquiry | Not reported in source |
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Procedure
Preparation of solutions before perfusion
Prepare the following two solutions. Perfusion prewash: add 1.25 ml of 1% (w/v) sodium nitrite and 0.04 ml of heparin (5000 IU/ml) to 100 ml of 0.9% (w/v) sodium chloride solution; prepare immediately before use. Perfusion fixative: 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). Prepare no more than 48 hours before use, store at 4°C, and bring to room temperature on the day of perfusion.
Induction of anesthesia
Induce anesthesia by intraperitoneal injection of ketamine (150 mg/kg) and xylazine (15 mg/kg).
Thoracotomy and left ventricular cannulation
Open the chest and insert a needle into the left ventricle. Connect the needle to tubing, with a T-connector linking the prewash and fixative solutions, and to a peristaltic pump set to 18 ml/min. Make a small incision in the right atrium to allow blood and perfusate to drain throughout the procedure.
Labeling of the arterial and capillary systems
If specific labeling of the arterial and capillary systems is required, perform this step immediately after opening the chest and exposing the heart: use a 31G needle to inject 100 µl of DyLight 649-conjugated tomato lectin (Lycopersicon esculentum, 1 mg/ml) directly into the left ventricle within 30 seconds. Allow the lectin to circulate for one minute before starting prewash perfusion.
Prewash perfusion
Perfuse with prewash solution until the right atrial outflow is clear and the liver and limbs are blanched (usually requiring approximately 18–20 ml of prewash solution).
Fixative perfusion
Perfuse with 90 ml of fixative. At completion, the organs should be firm and the tissues well preserved.
Tissue collection and postfixation
Dissect the tissues required for analysis and postfix them in fixative at 4°C for 18–24 hours.
PBS washing
Wash the tissues in phosphate-buffered saline (PBS; 0.1 M, pH 7.2) three times for 30 minutes each.
Storage
Store at 4°C in PBS containing 0.1% sodium azide until use for immunohistochemistry or other microscopic analyses.
Quality control and readouts
The prewash endpoint is clear outflow from the right atrium and blanching of the liver and limbs; the fixation endpoint is firm organs and well-preserved tissue. Prepare prewash solution immediately before use. Prepare fixative no more than 48 hours before use, store at 4°C, and bring to room temperature on the day of perfusion. Storage solution containing 0.1% sodium azide is incompatible with horseradish peroxidase (HRP)-based detection systems because sodium azide inhibits HRP.
The protocol produces well-preserved, perfusion-fixed adult mouse tissue (such as brain) for anatomical observation and subsequent microscopic analyses, including immunohistochemistry. Volume endpoints are approximately 18–20 mL of prewash solution until the outflow is clear and the liver and limbs are blanched, followed by 90 mL of fixative until the organs become firm.
References
- Keast JR, Bowden L, Osborne PB. Intracardiac perfusion of experimental mice with fixative for anatomical studies. protocols.io record 227096, v1 (published 2026-09-10). DOI:10.17504/protocols.io.bp2l6z7xdgqe · DOI: 10.17504/protocols.io.bp2l6z7xdgqe/v1
- Thermo Fisher Scientific, IHC Troubleshooting Guide (Pierce Protein Methods): Sodium azide inhibits HRP (sodium azide inhibits horseradish peroxidase activity)