Frozen Sectioning Protocol: Tissue Processing & Cryosectioning Guide

 

I. Tissue Processing for Frozen Sectioning

1. Fixation

The fixation method for frozen sectioning depends on the specific research objectives, but it generally falls into two categories: chemical fixation and cryofixation.

• Chemical Fixation: You must consider whether the fixative will interfere with the target structures, such as enzyme activity, expression, or immunoreactivity. Therefore, fixatives with lower cross-linking efficiency but better preservation of antigenicity or biochemical activity (e.g., low-concentration formalin) are typically preferred. Avoid high-efficiency fixatives like osmium tetroxide  or glutaraldehyde, as their intense cross-linking can destroy native tissue antigenicity.

• Cryofixation (Flash Freezing): Freshly harvested animal tissue is placed in a freezing embedding medium and directly immersed in liquid nitrogen or a coolant pre-cooled by liquid nitrogen (such as isopentane). This method provides the best preservation of biochemical and enzyme activities, making it ideal for enzymatic or immunological assays. However, post-sectioning storage requires stricter conditions to prevent the degradation of tissue structure and activity.

2. Embedding

Optimal Cutting Temperature (OCT compound) is commonly used as a matrix in frozen sectioning to provide adequate structural support for the tissue.

To prevent ice crystal formation—which causes uneven tissue hardness, structural damage, and hinders sectioning—fixed tissue blocks can be sequentially transferred into gradient sucrose buffer solutions (cryoprotection) before final embedding in OCT.

Key Rule: The faster the cooling process during embedding, the better the morphological preservation, and the lower the risk of ice crystal formation.

Rapid cooling can be achieved using liquid nitrogen or specialized coolants. Embedded tissue blocks can be stored in a freezer at -20°C to -70°C. One to two hours before sectioning, transfer the blocks from the freezer into the cryostat chamber to allow the tissue to acclimate to the optimal sectioning temperature.

If a partially sectioned tissue block needs to be archived, the exposed tissue surface must be resealed with OCT compound. This prevents water sublimation and evaporation from the cut surface, which causes tissue dehydration, shrinkage, and deformation. Embedded blocks should be sectioned as soon as possible. Although they can be stored long-term in an ultra-low temperature freezer (-20°C to -70°C), prolonged storage may lead to desiccation of the surface OCT, compromising the morphology and quality of future sections.

II. Cryosectioning Technique

1. Block Trimming

The embedded block appears white, while the tissue typically ranges from pale yellow-orange to reddish-brown. Since aluminum foil is often used as a mold during embedding, ensure it is completely removed before mounting the block to the chuck to avoid damaging the blade or the tissue.

Trimming should strictly aim to expose the target tissue surface. Simply shave away excess OCT compound that contains bubbles, debris, or irregular edges. Unlike paraffin sectioning, there is no need to trim the block into a trapezoidal pyramidal shape.

2. Operating the Cryostat

The operation and sectioning mechanics of a cryostat are similar to a paraffin microtome; however, close attention must be paid to the optimal sectioning temperature, and caution must be exercised to avoid frostbite or cuts.

Beyond adjusting the blade angle, proper positioning of the anti-roll plate is critical:

• Too far forward: It creates friction and compresses the tissue block, preventing smooth sectioning.

• Too far backward: The sections will curl up, making them impossible to flatten or mount onto slides.

• Correct position: The sections will roll out completely flat between the anti-roll plate and the blade.

Cryosectioning is typically performed one section at a time. Each section should be mounted onto a slide immediately after cutting, air-dried, and then frozen for storage. The chosen thickness depends on the experimental goals. Generally, frozen sections are thicker than paraffin sections, typically ranging from 10 to 30 μm.

3. Slide Mounting (Section Pick-up)

To mount a frozen section, take a pre-coated adhesive slide (adhesive side facing down) and gently touch it to the section resting on the blade. The relative warmth of the slide melts the section, causing it to adhere instantly.

Allow the mounted slides to air-dry naturally at room temperature before storage. Avoid touching the actual cutting edge of the blade with the slide to prevent blade contamination or damage.

If OCT residue builds up on the blade and hinders sectioning, gently wipe it away using gauze moistened with acetone. Wait for the blade to cool back down to the chamber temperature before resuming sectioning. After washing, the mounted sections can be stained with Hematoxylin & Eosin (H&E) or Toluidine Blue for morphological evaluation.

4. Storage of Sections

Frozen sections should be stored in slide boxes, sealed tightly, and kept at -20°C to -70°C to preserve tissue antigenicity and enzyme activity over long periods.