Key to Successful Pathological Staining! Core Techniques and Pitfall Avoidance Guide for Sample Preprocessing
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Pathological staining is a crucial link in disease diagnosis and scientific research. The standardization of sample preprocessing directly affects staining quality and result accuracy. This article provides a detailed analysis of the preprocessing steps for three commonly used sectioning techniques (fixed tissue frozen sectioning, fresh tissue frozen sectioning, and paraffin sectioning), offering references for scientific research and clinical practice.
1. Fixed Tissue Frozen Sectioning
Application Scenarios: Samples requiring long-term preservation or specific antigen retention.
Immediately immerse the tissue in 4% paraformaldehyde or 10% neutral formalin after excision. Adjust the fixation time according to the tissue size (usually 10-20 minutes to 24 hours). Short-term fixation is suitable for small samples, while large tissue blocks require extension to 24 hours to fully coagulate the protein structure.
After fixation, sequentially immerse the tissue in 15% → 30% sucrose solution (prepared with PBS), and let it stand overnight at each concentration until the tissue sinks to the bottom. Sucrose penetration and dehydration can reduce ice crystal formation and maintain cell morphology.
Place the dehydrated tissue on the cold stage of a cryostat, add OCT embedding medium to fully cover the tissue, and freeze rapidly at -20℃ 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 after mounting.
Post-Fixation and Permeabilization
Immerse the dried sections in 4% paraformaldehyde (4℃) for fixation for 10 minutes. After rinsing with PBS, permeabilize with 0.1% Triton X-100 for 5 minutes to enhance antibody penetration.
2. Fresh Tissue Frozen Sectioning
Application Scenarios: Intraoperative rapid pathological diagnosis or preservation of lipid/enzyme activity.
Immediately place the fresh tissue on the cold stage of a cryostat after excision, add OCT embedding medium, and freeze rapidly for 10 minutes. For temporary storage, freeze rapidly in liquid nitrogen and then store at -80℃.
Adjust the section thickness (8-10μm), mount the sections, and air-dry at room temperature for 30 minutes to ensure the sections adhere tightly to the glass slides.
Post-Fixation and Permeabilization
Immerse the dried sections in 4% paraformaldehyde (4℃) for fixation for 10 minutes. After rinsing with PBS, permeabilize with 0.1% Triton X-100 for 5 minutes to enhance antibody penetration.
Application Scenarios: High-resolution observation of tissue structure and long-term archiving.
Cut the tissue into 3-5mm thin blocks and fix in 4% paraformaldehyde for 12-24 hours. Large specimens (such as tumors) need to be extended to 24 hours for full penetration.
Gradually dehydrate with gradient ethanol (70% → 100%), 1-2 hours per grade; clear with xylene twice (15-30 minutes each time) to replace ethanol for paraffin infiltration.
Paraffin Infiltration and Embedding
Infiltrate the tissue with molten paraffin (60℃) for 2 hours, embed into blocks, and store temporarily at 4℃. Thorough paraffin infiltration is required to avoid section fragmentation.
Cut 4μm thin sections with a microtome and bake at 65℃ for 1-2 hours to enhance adhesion. After baking, deparaffinize with xylene (15 minutes × 2 times) and rehydrate to PBS with gradient ethanol.
1. Fixation Time: Fixation for less than 12 hours may lead to insufficient fixation, while more than 24 hours may affect antigen epitopes.
2. Dehydration Gradient: The concentration of ethanol or sucrose must be strictly increased incrementally to avoid tissue shrinkage and deformation.
3. OCT Embedding: The embedding medium must cover the entire circumference of the tissue to avoid air bubbles; the sectioning direction should be clearly marked.
4. Section Thickness: Frozen sections should be 8-10μm, and paraffin sections 4μm. Excessively thick sections are prone to detachment or uneven staining.
Through a standardized preprocessing workflow, tissue morphology and biomolecular activity can be preserved to the maximum extent, laying a solid foundation for subsequent HE staining, IHC, or mIHC. In practical operations, parameters should be flexibly adjusted according to the experimental purpose, and strictly refer to the reagent instructions and industry standards.
5. Brand Support from ANT BIO PTE. LTD.
To further ensure the success of pathological staining experiments, ANT BIO PTE. LTD. provides a full range of high-quality reagents and technical support through its Absin product line, including multiplex fluorescence IHC staining kits, IHC staining kits, and related preprocessing reagents (such as fixatives, dehydrating agents, and embedding media). All products have undergone strict quality control to match the standardized preprocessing steps described in this article, helping researchers avoid experimental pitfalls and improve experimental efficiency.
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This article is AI-compiled and interpreted based on the original work in document 1226.docx (Key to Successful Pathological Staining! Core Techniques and Pitfall Avoidance Guide for Sample Preprocessing). All technical information and product data are for research purposes only and are not intended for clinical diagnosis or treatment. For any product quality issues, please contact us promptly and we will handle them actively.
ANT BIO PTE. LTD. – Empowering Scientific Breakthroughs
At ANTBIO, we are committed to advancing life science research through high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) cover a full spectrum of research needs, from general reagents and kits to antibodies and recombinant proteins. With a focus on innovation, quality, and customer-centricity, we strive to be your trusted partner in unlocking scientific mysteries and driving medical progress. Explore our product portfolio today and elevate your research to new heights.
