Introduction

Animal sampling is an important part of animal experiments, and almost all animal experiments need sampling for further observation.

1. Pharmacological research: In order to evaluate the effects of drugs on specific organs (including but not limited to the cardiovascular, nervous, digestive, and respiratory systems, etc.) In pharmacological experiments, it is often necessary to take samples from the relevant tissues of experimental animals.
2. Toxicology research: In toxicology experiments, the extent and mechanism of damage caused by toxic substances to the body can be observed and analyzed through the sampling of various organs in the animal body.
3. Pathological research: In the field of pathology, tissue sampling is essential for the diagnosis and study of diseases. The sampling and analysis of pathological tissues can help to understand the pathogenesis and development of diseases.
4. Developmental biology and regenerative medicine research: In developmental biology and regenerative medicine research, tissue sampling helps track gene expression patterns during cell differentiation and development, as well as the ability to repair and regenerate.

Reagent preparation

• Anesthetics: inhaled anesthetics (isoflurane) or injectable anesthetics; small animal euthanasia equipment.
• Saline: used to wash tissue specimens
• Fixed solution: 10% neutral formaldehyde solution or 4%PFA, etc.

Equipment preparation

Animal fixation plate, anesthesia machine or small animal euthanasia device, blade, forceps (large, small, toothless tweezers, toothed tweezers), delicate scissors (large, small), hemostatic forceps, filter paper, gauze, ruler, fixed container (centrifugal tube, etc.), scalp needle, syringe, etc.

Anatomical sampling method

Animals should be anesthetized or euthanized quickly to avoid animals in a state of stress or near death for a long time, increases the suffering of animals and causes changes in tissues and cells in the body, and produces pathological illusions due to human interference, resulting in inaccuracy of diagnosis and affecting experimental results.

Taking rats and mice as an example, after the animals are anesthetized or euthanized, the animals are placed in a supine position and fixed in a fixation plate. Alcohol is used to wipe and sterilize the abdominal skin, and the need for shaving or hair removal can be assessed according to the needs of materials.

The subcutaneous muscle layer and the peritoneum layer were cut along the midabdominal line, and the sternum was cut upward to expose the thoracoabdominal organs.

Organ observation

After opening the chest and abdomen, observe the overall condition of the animal’s organs and tissues, and whether there are ascites or visible lesions. The target organ was removed according to the experimental purpose, and observed for the presence or absence of lesions and the shape, color, location, size, and sectioning of the lesions.

Anatomical announcements

• The organ tissue should be taken quickly, which can be operated on wet ice when sampling, and the tissue can be transferred to the fixation liquid as soon as possible.
• Try not to use toothed forceps to pick up the organ tissue, but use untoothed forceps to pick up the connective tissue around the organ to avoid artificial damage.
• The scissors and blades used for materials should be sharp.
• Larger tissues of the liver and kidney can be cut into chunks first and then cut to a suitable size after fixing them.
• Smaller tissues, such as the gland pituitary, should be flat on filter paper before fixation so that they can maintain a good shape after fixation.
• If there are blood, mucus and other contaminants on the organs, rinse with saline before fixing.
• The digestive system cavity organs are arranged in the order of stomach to rectum, separated along the mesentery, and affixed to the filter paper for fixation, or some intestines can be coiled into a “Swiss roll” for fixation.
• Flat tissues prone to curling, such as skin, can be spread flat on filter paper and placed in an embedding cassette for fixation.

Fixed method

To soak and fix, the removed tissue is soaked in the fixing solution. The fixed liquid must be sufficient, generally 10-50 times the total volume of the tissue block, and the fixed time is generally 48-72h to ensure that the fixed liquid fully penetrates into the tissue. If there is no time for dewatering and embedding after fixation, the organs can be temporarily stored in 70% ethanol, and long-term immersion in the fixing solution will affect the staining effect. The fixed container is placed on a low frequency oscillator for oscillation, or the fixed container is often manually shaken to accelerate the penetration rate of the fixed agent into the specimen, relatively shortening the fixed time, and making the specimen fixed penetration more uniform.

Tissue sampling
Kidneys, bladder:

• Left kidney: transversal incision is made from the renal hilum, and the incision clearly shows the renal hilum.
• Right kidney: Cut longitudinally along the whole kidney, including the renal hilum, to fully expose the renal pelvis.
• Bladder: The incision is made longitudinally with the bladder opening as the center, and the incision is perpendicular to the axis of the bladder.

Liver, spleen:

• Liver: Select the largest and smallest lobes and make a vertical incision at the portal of the liver, keeping the incision thickness at 3-5mm.
• Spleen: Perform a transverse incision along the leading edge of the spleen, ensuring that the incision passes through the main area of the spleen to facilitate observation of the internal structure of the spleen.

Lung, trachea:

• Lung: Select the left upper lobe and the right lower lobe, and make a vertical incision at the hilus of the lung, keeping the incision thickness at 3-5mm.
• Trachea: Transverse cutting. The length of the trachea is 3 to 5mm.

Stomach, duodenum, Peyer’s patches, jejunum:

• Stomach: Make a longitudinal incision from the cardia to the pylorus along the greater curvature of the stomach, keeping the incision thickness at 3-5mm.
• Duodenum and jejunum: Transverse incisions were made respectively, keeping the incision length between 3 and 5mm. The part containing Peyer’s patches was selected for the jejunum incision.

Ileum, cecum, colon, rectum:

Ileum, cecum, colon and rectum: Transverse incisions were made respectively, keeping the incision length within 3-5mm.

Thyroid gland, parathyroid gland:

Thyroid: Expose the parathyroid gland and make a longitudinal incision from behind the thyroid, keeping the incision thickness at 3-5mm.

Pituitary, pancreas:

• Pituitary gland: Make a transverse incision without trimming, and keep the incision thickness at 3-5mm.
• Pancreas: Perform a transverse incision, keeping the incision thickness at 3-5mm.

Testicles, epididymis:

Testis and epididymis: Make a transverse incision and keep the incision thickness at 3-5mm.

Salivary glands, submaxillary glands, submaxillary lymph nodes:

• Salivary gland and submandibular gland: longitudinal incision is made, keeping the incision thickness at 3-5mm.
• Submaxillary lymph nodes: only excess mucosa is removed, not pruned.

Skeletal muscle:

• Skeletal muscle: Make transverse and longitudinal incisions respectively, keeping the incision thickness at 3-5mm.

Sciatic nerve:

• Sciatic nerve: Transverse incision.

Heart, aorta:

• Heart: Make a longitudinal incision from the auricle, exposing the right atrium, atrioventricular valve, right ventricle, left atrium and other structures, and keep the incision thickness at 3-5mm.
• Aorta: Make a transverse incision, keeping the incision length between 3 and 5mm.

Prostate, seminal vesicles:

• Prostate and seminal vesicles: Make a transverse incision and keep the incision thickness at 3-5mm.

Tongue:

• Tongue: Make a transverse incision, remove the tip of the tongue, and keep the incision thickness at 3-5mm.

Skin:

• Skin: A 2x2x0.5cm skin sample is cut so that the epidermis, dermis and subcutaneous tissue structure of the skin can be observed.

Adrenal glands:

• Adrenal gland: Cut in half.

Eyeballs, optic nerves:

• Eyeball: Cut it in half, preserve the part with the optic nerve.

Brain and spinal cord:

• Brain: Transverse incisions were made in the forebrain, midbrain and cerebellum respectively, and the incision thickness was 3-5mm.
• Spinal cord: Transverse incisions were made in the back of the brain, thoracic vertebrae and lumbar vertebrae respectively, keeping the incision thickness at 3-5mm.

Thymus, mesenteric lymph nodes:

• Thymus: Make a transverse incision, keeping the incision length at 3-5mm.
• Mesenteric lymph nodes: only the excess mucosa is removed, not pruned.

After tissue fixation, paraffin sample blocks can be prepared by dehydration, transparency, embedding and other steps, and then sliced by RWD Rotary Microtome. Or after dehydration, it is embedded and sliced using RWD Cryostats. Then the stained samples were observed according to the experimental requirements.