Multi-Organ Collection from Mouse Models for Aging Studies

Aging is a systemic process that affects nearly all organs, yet most experimental studies focus on a single tissue, limiting insights into how age-related changes co-occur or interact across the body. Aging manifests in both tissue-specific and systemic alterations, including immune dysfunction, metabolic shifts, loss of regenerative capacity, and structural degeneration^1,2. While organ-specific investigations have provided valuable insights, a broader perspective is needed to understand how cellular aging across systems contributes to whole-organism decline. Multi-organ analysis in preclinical in vivo models, such as mice, is essential to identify coordinated structural, cellular, and molecular changes that contribute to age-associated diseases and loss of physiological resilience^3,4.

The laboratory mouse remains an essential and well-established preclinical model for aging research due to its genetic tractability, short lifespan, conserved aging pathways, and its ability to recapitulate key features of human aging, including immune dysfunction, tissue fibrosis, metabolic decline, and increased neoplasia^5,6,7. However, efficient and standardized multi-organ dissection protocols are not widely used. Such protocols are necessary to optimize histological comparisons, reduce animal usage, and integrate omics datasets across tissues from the same subject^8,9.

Here, we present a reproducible, time-efficient protocol for multi-organ perfusion, dissection, and preservation in mice. This protocol provides a step-by-step guide for the anatomical dissection and collection of major organs from young (2-3 months) and aged (20-24 months) mice, including the brain, eyes, thymus, heart, lungs, aorta, inguinal lymph nodes, gastrointestinal tract (esophagus, stomach, small intestine, colon), liver, pancreas, spleen, kidneys, bladder, adipose tissue, skeletal muscle, bone, and skin. Detailed instructions for tissue preservation compatible with histological, molecular, and cryosectioning workflows are included. This standardized approach enables researchers to assess organ-specific and systemic hallmarks of aging within the same experimental subjects, supporting integrated, high-quality aging research^4,10,11,12.

C57BL/6J mice were maintained at the University of California, San Francisco. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and overseen by the Laboratory Animal Resource Center. Mice were housed under a 12-h light-dark cycle, with fewer than five adult mice per cage. Euthanasia was performed in accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals¹³. For this protocol, young mice were sacrificed at 3 months, and aged mice at 22 months of age. Before proceeding with the dissection and organ collection, print and complete the preparation checklist (Table 1) and tissue collection checklist (Table 2). The reagents and the equipment used are listed in the Table of Materials.

1. Cervical dislocation

NOTE: Cervical dislocation is to be performed by a trained individual to minimize excessive trauma to the cervical structures.

1. Restrain the mouse by firmly grasping the base of the tail with one hand.
2. Place the thumb and forefinger of the opposite hand at the base of the skull.
3. Apply firm, rapid pressure to dislocate the cervical vertebrae. Palpate the neck to confirm separation between the skull and the adjacent vertebrae.
4. Confirm successful euthanasia (following institutionally approved protocols) by ensuring respiratory arrest and the absence of responses to noxious stimuli before proceeding.
5. Record the body weight of the mouse prior to perfusion and tissue collection.

2. Thoracotomy and perfusion

NOTE: Perfusion is optional. Perform all procedures involving paraformaldehyde (PFA) in a certified chemical fume hood while wearing appropriate personal protective equipment (PPE), including a lab coat, nitrile gloves, and safety glasses. Dispose of the PFA solutions in accordance with institutional chemical safety guidelines. Collect all waste PFA and contaminated materials (e.g., gloves, tissues, containers) in a clearly labeled hazardous waste container and dispose of them through the institution's Environmental Health & Safety office, following protocols compliant with local, state, and federal regulations.

CAUTION: If using paraformaldehyde (PFA) for fixation, perform all the steps under a fume hood to avoid exposure to formaldehyde vapors.

1. Place a styrofoam lid covered with an absorbent material to collect fluid from perfusion in a collection tray. Place the mouse on this dissection surface in a supine position and secure the limbs with pins to stabilize the body: forelimbs positioned laterally to open the thoracic cavity and hindlimbs extended caudally to create gentle tension across the abdominal wall.
   NOTE: This positioning improves visibility, minimizes obstruction, and maximizes working space for organ collection.
2. Apply 70% ethanol to the chest area.
3. Use sharp scissors to make a transverse incision at the level of the diaphragm, followed by two V-shaped cuts extending to expose the rib cage (Figure 1A).
4. Using blunt-end forceps, gently lift the skin and carefully cut any remaining attachments to fully expose the thoracic area.
5. Make two approximately 2 cm-long V-shaped incisions along both sides of the rib cage to open the thoracic cavity.
6. Pull up on the sternum and pin it over the shoulder of the mouse alongside the head.
7. Using blunt-end forceps, carefully expose the heart. Remove any trapped air bubbles from the perfusion line, then insert a butterfly needle or cannula into the tip of the left ventricle to a depth of approximately 3-4 mm. Make an approximately 5 mm-long incision in the right atrium to allow outflow of the perfusate (Figure 1B).
8. Begin perfusion with a Calcium- and Magnesium-free physiological solution (e.g., PBS, saline, or HBSS) to clear the circulatory system.
   NOTE: Perfusion can be performed using a 20 mL syringe, pump or gravity-fed system. On average, 15-20 mL of perfusion solution is required per mouse to achieve complete clearance of blood from the vasculature. Good perfusion is indicated by rapid clearing (blanching) of the liver and lungs, as well as the mouth area and tongue appearing pale. Complete perfusion will result in clear, blood-free outflow from the heart.
9. If desired, initiate PFA perfusion by switching the perfusion line from calcium- and magnesium-free physiological solution (used for pre-flushing and blood removal) to a 50 mL syringe containing 4% paraformaldehyde (PFA), while keeping the needle in place. Take care to avoid introducing air bubbles into the line. Continue perfusing with 4% PFA until the desired degree of fixation is judged by the progressive stiffening of the body. Approximately 30 mL of fixative is sufficient for a single mouse.

3. Organ and tissue collection

NOTE: The sequence of organ collection is critical when comparing young and aged mice. Organs in aged animals tend to be fragile and susceptible to autolysis or mechanical damage, making prompt and consistent tissue handling essential. Standardizing the order of collection minimizes postmortem variability, reduces handling artifacts, and ensures uniform fixation quality across animals^14,15. Consistency is vital for detecting subtle age-related morphological changes, as delayed or inconsistent sampling can introduce bias. If PFA perfusion has not been performed, organs should be harvested rapidly. The pancreas, stomach, lungs, and brain are particularly prone to rapid autolysis and histological degradation if not processed immediately. Because aged mice frequently develop neoplastic lesions, examine the spleen, liver, lymph nodes, lungs, and thymus carefully for asymmetry, enlargement, nodules, or discoloration. Record these findings as part of the overall disease burden (see Table 2).

1. Begin with the animal in a supine position following perfusion or cervical dislocation to collect the major organs of the thoracic and abdominal cavities (Figure 1C).
2. Gently apply 70% ethanol using gauze or a spray bottle after pinning, which minimizes fur contamination and maintains tissue hydration in sensitive regions. This method has proven effective in our hands for reducing fur debris and improving visibility during dissection while avoiding the drawbacks of full immersion.
3. Using scissors and blunt-end forceps, carefully open the abdominal cavity by making a midline incision from the diaphragm to the pelvis, followed by two lateral incisions as indicated by the red lines in Figure 1A. Pull up the skin and abdominal wall to fully expose the internal organs.
4. Stomach, small intestine, and colon: Detach the stomach at the esophageal junction while leaving the pyloric end continuous with the small intestine. Gently free the gastrointestinal tract by severing the supporting ligaments, then remove the tract, including the small intestine and colon. Cut distally at the rectum.
   1. To clear luminal contents, sever the small intestine proximally at the duodenojejunal flexure and distally at the ileocecal junction. For the colon, sever proximally at the cecum. Flush the small intestine and colon with approximately 20 mL of physiological solution using a syringe fitted with a feeding needle, while gently securing the tissue with fine-tip forceps to avoid damage.
5. Esophagus: Isolate the esophagus from the cervical to the gastric junction.
6. Spleen and pancreas: Isolate the spleen and pancreas from surrounding tissues using fine dissection, minimizing mechanical damage. Carefully detach the pancreas from the spleen. Immediately dissect and trim the pancreas into small pieces (≤5 mm) and immerse them in fixative within 5 min of euthanasia to minimize autolysis and preserve optimal tissue morphology. Inspect the spleen, noting size, color, shape, and the presence of any visible abnormalities.
7. Liver: Using fine tip forceps, grasp the central connective tissue between the liver lobes and gently pull upward to create tension. With dissection scissors, carefully sever all attachments between the liver and adjacent organs. Inspect the liver for color, consistency, and the presence of nodules or other abnormalities.
8. Visceral fat: Collect samples of visceral adipose depots.
   NOTE: Young animals typically have minimal visceral fat, whereas aged animals often exhibit increased fat accumulation, particularly around the intestines, kidneys, and gonadal regions.
9. Kidneys and bladder: Excise both kidneys along with the attached ureters and bladder. Carefully detach the kidneys from the surrounding adipose tissue and connective structures using fine scissors or blunt-end forceps. Follow the ureters distally to the bladder, cutting any remaining attachments to isolate the entire urinary tract. Inspect the kidneys and bladder for size, color, and abnormalities before further processing.
10. Lymph nodes: Retract the skin from the peritoneum to expose the inguinal region. Identify the lymph nodes within the mammary fat pad along the lymphatic vessels. Isolate and remove the nodes with fine-tip forceps, minimizing disruption of surrounding fat and connective tissue.
11. Heart: Carefully remove the heart by severing the major vessels.
12. Thymus: Excise the thymus carefully to preserve its structure. Be aware that the thymus undergoes involution with age, and in aged mice, only minimal or no thymic tissue may be present.
13. Lungs: Using sharp scissors and fine-tip forceps, carefully remove the lungs along with the attached trachea in one piece.
14. Aorta: Insert closed forceps between the aorta and the spine and gently dislodge the aorta, separating it along the dorsal aspect. Take care to avoid tearing the vessel. Using fine-tip forceps, carefully remove surrounding adipose and connective tissue from the aorta under a dissection microscope.
15. Skeletal muscle: Resect the skin over the hindlimbs to expose the femur and surrounding musculature. Using fine scissors, carefully isolate the muscles attached to the femur by detaching them from surrounding connective tissue and severing at the tendon insertions to preserve fiber integrity.
16. Bone (Tibia): Using dissection scissors, resect the overlying skin and muscle to expose the tibia. Using dissection scissors and blunt forceps, carefully detach the tibia at the knee and ankle joints. Remove any remaining soft tissue without damaging the bone.
17. Eyes: Using large dissection scissors, decapitate the mouse. Carefully remove the eyes using fine-tip forceps.
18. Brain: Resect the scalp to expose the anterior skull. Using sharp scissors or a scalpel, follow the cranial sutures and lift away the bone segments to expose the brain (Figure 1D). Sever the olfactory bulbs and ventral attachments. Gently extract the brain from the skull cavity. Process the tissue immediately to maintain morphology.
19. Skin: If hair removal is required for downstream applications, remove hair using an electric trimmer. Pinch a portion of the back skin with blunt forceps, excise with scissors, and collect the tissue for further processing.
20. After removing all organs of interest, place the entire mouse carcass in a 450 mL container pre-filled with 4% PFA to archive for unexpected findings or to answer reviewers' questions that may require return to wet-fixed tissue.

4. Tissue preservation and processing

NOTE: To preserve tissue integrity, minimize the time between dissection and preservation. Delays can compromise tissue morphology and molecular quality; further details for critical timing of tissue collection and preservation are shown in Table 2.

CAUTION: If using paraformaldehyde (PFA) for fixation, perform all steps inside a fume hood to avoid exposure to formaldehyde vapors.

1. Process tissues immediately after dissection, selecting the appropriate protocol (step 4.1, 4.2, or 4.3) according to the intended downstream application.
   1. Paraformaldehyde (PFA) fixation: Place tissues intended for histological analysis in 4% PFA at 4 °C for 12-24 h. Following fixation, wash tissues thoroughly with phosphate-buffered saline (PBS) to remove residual fixative, then transfer them to 70% ethanol for storage until paraffin embedding and sectioning.
2. Optimal Cutting Temperature (OCT) compound embedding: Embed fresh tissues in OCT compound. Place tissues in embedding molds filled with OCT compound, eliminating air bubbles, incubate for 5 min at room temperature, then freeze in the vapor phase of liquid nitrogen. Seal with aluminum foil and store OCT blocks at −80 °C until sectioning.
3. Snap freezing in liquid nitrogen vapor phase: Snap-freeze tissues intended for RNA, protein, or metabolite analysis in the vapor phase of liquid nitrogen to minimize ice crystal formation. Place tissues in labeled cryotubes and suspend them above liquid nitrogen for rapid freezing. Once fully frozen, transfer samples to −80 °C storage.

During necropsy, aged mice frequently display neoplastic lesions and require careful examination to document pathological findings13,14,16,17,18. Figure 2A shows a 22-month-old male C57BL/6 mouse in the supine position following perfusion. Complete blanching of the lungs confirms effective perfusion, while the liver exhibits only partial blanching. Closer inspection of the liver revealed multiple malignant nodules (Figure 2B), accompanied by enlarged seminal vesicles (Figure 2A) and splenomegaly (Figure 2C). These findings are common in aged mice and should be documented as part of the total disease burden18.

Figure 3 depicts macroscopically normal organs collected following dissection and perfusion, allowing assessment of anatomical integrity and perfusion efficiency. The gastrointestinal tract was flushed with physiological solution to remove digested contents, resulting in a clean, translucent appearance of the stomach, colon, and small intestine (Figure 3A). Additional structures, including the esophagus, pancreas, spleen, visceral adipose depots, kidneys with bladder, lymph nodes, heart, and thymus, were clearly identifiable and could be isolated intact for downstream analyses (Figure 3B,C,E-I).

Perfusion quality was evaluated by examining blanching in highly vascularized tissues. As shown in Figure 3D and Figure3J, the liver and lungs displayed distinct differences between non-perfused, well-perfused, and partially perfused conditions. Complete blanching confirmed successful perfusion, whereas incomplete blanching indicated technical variability or underlying abnormalities. Other tissues, including aorta, skeletal muscle, tibia, brain, eye, and skin, were isolated with preserved morphology, suitable for further histological, molecular, or imaging applications (Figure 3K-P). This standardized dissection procedure provides consistent recovery of intact, well-preserved tissues and organs and enables gross anatomical assessment to support a variety of downstream analyses. Dissection and organ collection required approximately 40-60 min per animal, depending on operator experience and the presence of pathological findings.

Figure 4 shows exemplar aging-associated changes in collected organs following paraffin embedding, sectioning, and hematoxylin and eosin (H&E) staining. In skin sections from aged mice, epithelial thinning and loss of hair follicles were observed compared to young controls (Figure 4A). Kidney sections revealed enlargement of Bowman's capsules along with fibrotic and necrotic changes, as well as regions consistent with lymphomas (Figure 4B). In pancreatic sections, an increase in apoptotic bodies19 and lymphomatous regions was observed in aged animals, indicative of compromised tissue integrity with age (Figure 4C). Spleen sections depicted amyloid deposits and lymphoma regions in aged animals, reflecting additional hallmarks of age-related pathology (Figure 4D).

These results highlight representative histopathological features of aging across multiple tissues, demonstrating the utility of this protocol for detecting structural hallmarks of age-related decline.

Figure 1: Schematic overview of mouse dissection and organ exposure procedures. (A) Incision sites for opening the thoracic and abdominal cavities (red lines), including a transverse incision at the level of the diaphragm, followed by V-shaped cuts along both sides of the rib cage to open the thoracic cavity, and a midline incision from the diaphragm to the pelvis with bilateral cuts to fully expose the abdominal cavity. (B) Perfusion setup showing insertion of a butterfly needle into the left ventricle and incision of the right atrium to enable effective blood drainage. (C) Anatomical layout of major thoracic and abdominal organs following cavity exposure. (D) Head dissection steps, including removal of the scalp and opening of the skull along the cranial sutures to expose the brain. Red lines indicate incision sites. Please click here to view a larger version of this figure.

Figure 2: Identification of neoplastic lesions in aged mice. (A) Representative 22-month-old male C57BL/6 mouse in the supine position following perfusion with 4% PFA. Complete blanching of the lungs (pink arrowhead) confirms effective perfusion, whereas the liver (green arrowhead) displays only partial blanching. Blue arrowheads denote enlarged seminal vesicles.(B) Detailed view of the liver revealing multiple malignant nodules (black arrowheads). (C) Enlarged spleen from an aged C57BL/6 mouse (right-hand side) in comparison to a normal spleen from a young animal (left-hand side). Please click here to view a larger version of this figure.

Figure 3: Overview of major mouse organs and tissue integrity following dissection and perfusion. (A) Stomach, small intestine, and colon before and after flushing with physiological solution to remove digested contents.(B) Esophagus.(C) Pancreas with attached spleen.(D) Liver without perfusion, with complete blanching after effective perfusion, and with partial blanching indicative of incomplete perfusion or underlying pathology.(E) Visceral adipose tissue.(F) Kidneys with attached bladder. (G) Inguinal lymph nodes.(H) Heart.(I) Thymus.(J) Lungs without perfusion, with complete blanching after effective perfusion, and with partial blanching.(K) Aorta.(L) Skeletal muscle isolated from the hindlimb.(M) Tibia with preserved bone structure.(N) Brain.(O) Eye.(P) Skin.Scale bars: 5 mm in A, C, D, E, F, J, L, N, P; 1 mm in all other panels. Please click here to view a larger version of this figure.

Figure 4: Histological analysis of major organs using hematoxylin and eosin (H&E) staining. (A) Skin sections from 3-month-old (young) and 22-month-old (aged) mice. Sections from aged animals show epithelial thinning and loss of hair follicles.(B) Kidney sections highlighting Bowman's capsules. Sections from aged animals show capsule enlargement, fibrotic and necrotic changes, and lymphomas (orange arrowheads).(C) Pancreatic sections from young and aged animals, with aged animals showing increased apoptotic bodies (blue arrowheads) and lymphoma regions (orange arrowheads).(D) Spleen sections from 3-month-old (young) and 22-month-old (aged) mice. Sections from aged animals show amyloid deposits (lime-green arrowheads) and lymphoma regions.Scale bar (A-C) 100 µm. Scale bar (D) 500 µm. Please click here to view a larger version of this figure.

Table 1: Preparation checklist. A step-by-step list of essential preparations for mouse tissue dissection and preservation, including tube and mold labeling, buffer and fixative preparation, setup for cryo-embedding, dissection tool sterilization, workstation arrangement, and documentation planning. Please click here to download this Table.

Table 2: Tissue collection checklist. Use the table to log collected tissues. Add time of collection and notes on condition or abnormalities. Prioritize the tissues critical for your study; collect the most relevant organs within the recommended timespan to avoid tissue autolysis/degradation.*Note any abnormalities (including organ discoloration, nodules, malignant growth, abnormal organ size). **Consider documenting preservation method (including PFA fixation, OCT embedding, or snap freezing). Please click here to download this Table.

This protocol provides a reproducible strategy for the systematic collection of multiple organ systems in young and aged mice, enabling anatomical, histological, and molecular analyses. Several technical considerations may affect outcome quality and address limitations of the described method. First, variability in perfusion quality is an important factor to consider. Incomplete perfusion may leave residual blood that compromises histology, immunohistochemistry, and molecular analyses. Perfusion after cervical dislocation can cause trauma to cervical tissues and lead to blood contamination of the lungs. Cervical dislocation should therefore only be performed by trained personnel. For optimal results, perfusion should be performed under terminal anesthesia as described elsewhere^20,21 in accordance with IACUC and AVMA guidelines¹³. Second, fixation is a critical determinant of tissue integrity. Although 4% paraformaldehyde (PFA) is widely used, it may inadequately penetrate large or dense organs, resulting in poor histology and antigen preservation. This limitation can be mitigated by using 10% neutral buffered formalin (NBF) or slicing tissues into ≤5 mm sections to improve penetration²². Third, some tissues, particularly pancreas, stomach, and brain, undergo rapid autolysis, necessitating fixation or freezing within 5-10 min of euthanasia and the use of pre-chilled dissection tools²³. In aged mice, neoplasia may further distort anatomy and complicate dissection. Additional artifacts may arise from inadequate fixation volume or freezing, leading to shrinkage and edge effects; these can be minimized by maintaining a fixative-to-tissue ratio of at least 10:1, avoiding overcrowding, and pre-cooling OCT molds before embedding. Finally, the use of PFA and formalin poses safety and disposal concerns; glyoxal-based fixatives offer less toxic alternatives. Standardized handling, optimized fixation, and careful timing are therefore essential for maximizing reproducibility, tissue quality, and the reliability of downstream analyses²⁴.

By harvesting tissues from over 15 organs within the same animal, this workflow maximizes biological yield per subject and aligns with ethical mandates to reduce animal usage. The systematic procedure can be readily scaled up to medium-throughput studies examining age-related physiological decline and organ-specific pathologies²⁵.

Prompt processing of autolysis-prone tissues is essential to preserve histological integrity. The use of calcium- and magnesium-free physiological solutions, followed by 4% PFA, reliably preserves organ architecture and minimizes autolysis in sensitive tissues like the pancreas and digestive system. Unlike protocols focused on single organs or disease models, this approach enables rapid and consistent collection of multiple organs, reducing variability and improving throughput. The entire dissection, from perfusion to final tissue excision, can be completed within 60 min per animal, making it suitable for large-scale or time-sensitive studies^3,11,26. Adherence to a precise perfusion-fixation sequence minimizes artifacts and enables reproducible outcomes.

Consistency in tissue collection, trimming, and embedding is essential for accurate histopathological assessment, particularly in longitudinal or multi-center studies. Variability in how tissues are handled can introduce artifacts, obscure pathological findings, or lead to misinterpretation of disease burden. The Revised Guides for Organ Sampling and Trimming in Rats and Mice, such as the RENI trim guides, provide detailed, standardized protocols to minimize such inconsistencies²². Adoption of these harmonized methods improves data comparability across studies and institutions, strengthens reproducibility, and enhances the integrity of pathology-based outcomes in aging research^4,9.

Collecting a multitude of organs is particularly important in aging research, where understanding how organ systems interact is essential to uncover the processes that drive whole-body physiological decline with age. While this approach can also be beneficial for a variety of other research questions, the focus of this protocol is to support aging studies where age-related changes may affect multiple organ systems simultaneously. However, it can also be applied to studies focusing on fibrosis, regeneration, and senescence, especially those integrating transcriptomics or multi-tissue biomarker discovery^9,27,28.

Histological assessments revealed characteristic age-associated alterations across multiple tissues (Figure 4). In skin, aged animals exhibited epithelial thinning and loss of hair follicles. In the kidney, sections from aged mice demonstrated Bowman's capsule enlargement along with fibrotic, necrotic, and lymphomatous changes. Pancreatic tissue from aged animals showed an accumulation of apoptotic bodies and lymphomas, while spleen sections revealed amyloid deposits and additional lymphoma regions. These observations are consistent with established murine aging phenotypes and demonstrate the utility of this protocol for detecting representative histopathological markers of aging^{1,18,19,27,29,30,31,32,33,34}.

In summary, this protocol offers a reliable, scalable framework for collecting and preserving tissues in preclinical aging models. It can be adopted in both exploratory and hypothesis-driven studies to uncover tissue-specific and systemic mechanisms of aging, degeneration, and resilience.