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The Key to Successful Histopathological Staining! Essential Techniques and Pitfall Avoidance in Sample Preprocessing
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一、Fixed Tissue Cryosection Applicable Scenarios: Samples that require long-term preservation or specific antigen preservation. Step-by-Step Details： Fixation:
Immediately after excision, immerse the tissue in 4% polyformaldehyde or 10% neutral formalin. The fixation time is adjusted according to the size of the tissue (usually 10-20 minutes to 24 hours). Short-term fixation is suitable for small samples, while larger tissues require an extension up to 24 hours for adequate protein structure solidification.
Gradient Sucrose Dehydration:
After fixation, immerse the tissue sequentially in 15% → 30% sucrose solutions (prepared with PBS), leaving it overnight at each concentration until the tissue sinks to the bottom. Sucrose infiltration dehydration can reduce ice crystal formation and maintain cellular morphology.
OCT Embedding and Sectioning:
After dehydration, place the tissue on the cold stage of the cryosection machine, add OCT embedding agent to fully cover, and quickly freeze at -20°C for 10-20 minutes. After embedding, trim the block, adjust the section thickness (usually 8-10µm), and air-dry the sections at room temperature for later use.
Post-fixation and Permeabilization:
Immerse the dried sections in 4% polyformaldehyde (4°C) for 10 minutes, rinse with PBS, then permeabilize with 0.1% Triton X-100 for 5 minutes to enhance antibody penetration.
二、Fresh Tissue Cryosection Applicable Scenarios：Rapid intraoperative pathological diagnosis or preservation of lipids/enzyme activity. Step-by-Step Details： Quick Freezing and Embedding:
Immediately after excision, place the fresh tissue on the cold stage of the cryosection machine, add OCT embedding agent, and quickly freeze for 10 minutes. If temporary storage is needed, quickly freeze with liquid nitrogen and store at -80°C.
Sectioning and Drying:
Adjust the section thickness (8-10µm), place the sections, and air-dry at room temperature for 30 minutes to ensure the sections adhere tightly to the glass slide.
Post-fixation and Permeabilization:
Immerse the dried sections in 4% polyformaldehyde (4°C) for 10 minutes, rinse with PBS, then permeabilize with 0.1% Triton X-100 for 5 minutes to enhance antibody penetration.
三、Paraffin Section Applicable Scenarios: High-resolution tissue structure observation and long-term archiving. Step-by-Step Details： Fixation:
Cut the tissue into 3-5mm thick blocks and fix with 4% polyformaldehyde for 12-24 hours. Larger specimens (such as tumors) require an extension up to 24 hours for adequate penetration.
Dehydration and Clarification:
Gradually dehydrate with ethanol (70% → 100%) for 1-2 hours at each step; clarify with xylene twice (each 15-30 minutes) to replace ethanol and facilitate paraffin infiltration.
Infiltration and Embedding:
Infiltrate the tissue with molten paraffin (60°C) for 2 hours, embed into blocks, and temporarily store at 4°C. Thorough infiltration is necessary to prevent section cracking.
Sectioning and Baking:
Use a microtome to cut 4µm thin sections, and bake at 65°C for 1-2 hours to enhance adhesion. After baking, dewax with xylene (15 minutes × 2 times), and rehydrate through a gradient ethanol series to PBS.
Key Considerations Fixation Time: Less than 12 hours may result in insufficient fixation, while more than 24 hours may affect antigen epitopes.
Dehydration Gradient: The concentration of ethanol or sucrose must be strictly increased to avoid tissue contraction and deformation.
OCT Embedding:
The embedding agent must fully cover the tissue to avoid air bubbles; the sectioning direction should be clearly marked.
Section Thickness:
Cryosections should be 8-10µm, and paraffin sections 4µm; excessively thick sections are prone to detachment or uneven staining.
By standardizing the pre-treatment process, you can maximize the preservation of tissue morphology and biomolecular activity, laying the foundation for subsequent HE staining, IHC, or mIHC. In practice, parameters should be flexibly adjusted according to the purpose of the experiment and strictly follow the instructions and industry standards.
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June 11, 2025
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