Preprocessing and Precautions for Cryosectioning

In clinical settings, cryosectioning is mainly used for rapid intraoperative pathological diagnosis, where the tissue removed during surgery is directly cryosectioned and then quickly stained, usually without preprocessing.

In research settings, modeled mice and rats often undergo perfusion fixation, tissue collection, and gradient dehydration before being preserved for cryosectioning. The purpose of perfusion fixation is mainly to cross-link protein and nucleic acid molecules to preserve the morphology of cells and tissues, to prevent lysosomal damage that leads to cell rupture, and microbiological degradation.

Processing Procedure

Perfusion (Using Mouse Brain Tissue as an Example)

Purpose: To deliver fixative through the vascular system to various organs for rapid fixation. Small animals are usually perfused via the heart, while arterial perfusion is recommended for larger animals.

Perfusion Solutions: Saline/PBS, 4% Paraformaldehyde (PFA).

Equipment Used: RWD TAIJI anesthesia machine, surgical operating table (or foam board), ophthalmic scissors, dissection forceps, hemostat, disposable infusion set, saline injection bottle, PFA injection bottle, etc.

• 1) Anesthetize the mouse using the RWD TAIJI anesthesia machine, fix it on the small animal surgical operating table with the abdomen facing up, and hang the saline injection bottle and PFA injection bottle on the infusion rack or infusion pole.

• 2) Make a lateral skin incision at the lower edge of the mouse’s xiphoid to expose the xiphoid, and cut along both sides to expose the diaphragm. Continue cutting the skin upward to expose the ribcage. Then extend the incision downward to expose the liver to observe the perfusion effect.

• 3) Cut the diaphragm below the xiphoid. Then use scissors to cut the ribs on both sides toward the head to expose the lungs and heart apex.

• 4) Use hemostat to lift the xiphoid upward, exposing the lungs, heart, and thymus. Fix the holding forceps on the operating table to prevent movement.

• 5) Insert the infusion needle of the disposable infusion set into the left ventricle (slightly left and upward from the apex). Use a hemostat to clamp the infusion needle inside the left ventricle to prevent it from falling off and fix the hemostat to the operating table to avoid movement. Connect the plastic needle of the infusion set to the saline injection bottle and tighten the flow regulator.

• 6) Cut the right pericardial sac and open the flow regulator to let saline enter the bloodstream. The blood and circulated saline will be expelled through the cut. Stop the saline perfusion when the liver turns completely pale.

• 7) Switch the perfusion solution from saline to 4% PFA. During the perfusion process, the mouse limbs shake and the tail wags. When the mouse is stiff all over the body, stop the PFA perfusion.

Tissue Collection

Purpose: To collect tissue for dehydration and sectioning.

Instruments Used: Curved dissection forceps, dissection scissors, ophthalmic scissors, etc.

• 1) Use dissection scissors to cut off the head of the perfused and fixed mouse.

• 2) Use ophthalmic scissors to cut the skin from the occipital end to the nasal end of the mouse’s head, exposing the cranial bones. The coronal suture, sagittal suture, Bregma, and Lambda points should be visible at this stage.

• 3) Use ophthalmic scissors to cut along the sagittal suture of the cranial bones. Ensure that the tip of the scissors is pressed against the bone when inserted from the occipital end to avoid damaging the cerebral cortex. Then use dissection forceps or the back of the ophthalmic scissors to pry open the cranial bones along the sagittal suture.

• 4) Use curved hook forceps to gently and slowly separate the brain tissue from the cranial bones, and cut the cranial nerves at the base. Remove the olfactory bulbs at the anterior end of the brain if necessary. Take care not to damage the cerebral cortex with the curved hook forceps and avoid pulling on the meninges. Once the brain tissue is separated, immerse it in PBS and place it on ice.

Dehydration

Purpose: To reduce ice crystal formation during tissue freezing, the different concentrations of sucrose are used to remove water from brain tissues via osmotic pressure.

Dehydrating solutions: 20%, 25%, 30% sucrose in PBS.

• 1) Dehydration Procedure: Immerse the brain tissue in the 20% sucrose solution. Once it sinks, replace the solution with higher-concentration sucrose solution and store it in a 4°C refrigerator until it sinks again.