Preparation of Tissue Sections in Histopathology

Ms. Sangeeta Gourh

Introduction

  • The preparation of tissue sections is an essential step in histopathology.
  • Different techniques are used depending on whether we want to study gross tissue architecture, cellular details, cytology, enzyme activity, or ultrastructure.

Paraffin Wax Embedding Method 

  • Most widely used method in diagnostic histopathology.

  • Produces thin, high-quality sections suitable for staining and long-term storage.

Steps:

  1. Fixation

    • Prevents autolysis and putrefaction.

    • Common fixative: 10% Neutral Buffered Formalin (NBF).

    • Fixation time: 6–48 hours (depending on tissue size).

  2. Dehydration

    • Tissue water is removed using increasing grades of alcohol (70%, 80%, 90%, 100%).

  3. Clearing

    • Replaces alcohol with a clearing agent (xylene, chloroform, or toluene).

    • Makes tissue transparent and miscible with paraffin.

  4. Infiltration

    • Tissue is impregnated with molten paraffin wax (56–60°C).

  5. Embedding

    • Tissue is oriented properly in a wax block.

    • Block is cooled and solidified.

  6. Section Cutting

    • Done with a rotary microtome.

    • Typical thickness: 3–5 µm for light microscopy.

  7. Staining

    • Routine: Hematoxylin & Eosin (H&E).

    • Special stains: PAS, Masson’s Trichrome, Reticulin, etc.

  8. Mounting

    • Sections are mounted with DPX or Canada balsam and covered with a coverslip.

✔ Advantages: Excellent preservation of morphology, permanent slides.
✘ Disadvantages: Slow process (12–24 hrs), lipids and some antigens may be lost.

Frozen Section Method

  • Used when rapid intraoperative diagnosis is required (oncology, margins of excision).

  • Preserves lipids, enzymes, and antigens better than paraffin.

Steps:

  1. Fresh tissue rapidly frozen using liquid nitrogen, isopentane, or cryostat refrigerant.

  2. Sectioned using a cryostat microtome at −20°C.

  3. Sections mounted on glass slides.

  4. Stained quickly with rapid H&E, Oil Red O (for fat), or enzyme histochemistry.

✔ Advantages: Very fast (results in 10–15 min), useful for enzyme/lipid studies.
✘ Disadvantages: Poorer section quality, cannot be preserved for long periods.

Cell Block Technique

  • Special method for cytology specimens (body fluids, FNAC aspirates).

  • Converts dispersed cells into a tissue-like block.

Steps:

  1. Centrifugation of sample.

  2. Formation of a cell clot (by adding plasma + thrombin, or by natural sedimentation).

  3. Fixed in formalin and processed like paraffin blocks.

  4. Sections cut and stained.

✔ Advantages: Preserves cell architecture, multiple stains possible.
✘ Disadvantages: Requires good cellularity.

Resin Embedding Method

  • Used in electron microscopy (EM) and sometimes for very hard tissues (e.g., bone, teeth).

Steps:

  1. Fixation: Glutaraldehyde (primary) + Osmium tetroxide (secondary).

  2. Dehydration: Acetone or alcohol.

  3. Infiltration: With epoxy resins (Epon, Araldite) or acrylic resins.

  4. Polymerization: Resin block hardened by heat or UV light.

  5. Ultramicrotome used for sectioning (50–100 nm thickness).

  6. Staining with heavy metals (uranyl acetate, lead citrate) for EM contrast.

✔ Advantages: Preserves ultrastructure of organelles.
✘ Disadvantages: Expensive, requires specialized setup.

Cell Smear / Impression Cytology

  • Used for cytology and quick diagnosis.

  • Types:

    • Touch Preparation (Imprint Smear): Tissue surface pressed onto a slide.

    • Crush Smear: Tissue gently crushed between two slides.

    • Scrape Smear: Tissue scraped and spread on slide.

✔ Advantages: Simple, quick, inexpensive.
✘ Disadvantages: No tissue architecture preserved, only cytological details.

Whole Mount Sections

  • Small specimens (embryos, insects, worms) are embedded and sectioned in entirety.

  • Useful in developmental biology, parasitology, embryology.

✔ Advantages: Study of the entire structure.
✘ Disadvantages: Only small specimens can be used.

Free-Hand Section 

  • Oldest method, still used in botany labs and basic teaching.

  • Fresh or fixed tissue cut manually with a razor blade.

✔ Advantages: No equipment required, very quick.
✘ Disadvantages: Uneven sections, poor detail.

Special Embedding Techniques

  • Celloidin Embedding

    • Uses celloidin (a form of nitrocellulose).

    • Good for brain, eye, and delicate tissues.

    • Provides support, avoids shrinkage.

  • Double Embedding (Paraffin + Celloidin)

    • Used for hard tissues like bone.

  • Gelatin Embedding

    • For frozen sections, particularly muscle and nerve tissues.

 

Comparison Table

Method Section Thickness Time Required Preservation Best For
Paraffin Wax 3–5 µm 12–24 hrs Good Routine histology
Frozen Section 8–12 µm 10–20 min Moderate Rapid diagnosis, lipids
Resin Embedding 50–100 nm 2–7 days Excellent Electron microscopy
Cell Block 3–5 µm 1 day Good Cytology samples
Smear/Imprint — Immediate Fair Cytology
Free-Hand Section 20–100 µm Immediate Poor Teaching, botany