Tissue preparation and staining

Tissue preparation and staining

Tissue preparation and staining

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Fixation - Pinning Tissues Down

  • Primary Goal: Halt autolysis (enzyme self-digestion) and putrefaction (bacterial decay) to preserve tissue architecture in a life-like state.
  • Mechanism: Inactivates enzymes and cross-links proteins, making them insoluble.
  • Routine Fixative: 10% neutral buffered formalin.
    • This is a 4% formaldehyde solution that forms methylene bridges between proteins.
    • Excellent for light microscopy and immunohistochemistry (IHC).
  • Specialized Fixatives:
    • Glutaraldehyde: For electron microscopy; offers superior ultrastructural preservation.
    • Alcohol: For cytologic smears; works by dehydration and protein denaturation.

⭐ Formalin fixation is reversible with heat, a key principle behind antigen retrieval techniques used in IHC to unmask epitopes.

Formaldehyde cross-linking mechanisms in tissue fixation

Processing & Sectioning - The Slice of Life

  • Goal: Transform fixed, soft tissue into a solid block that can be sliced into microscopically thin sections.
  • Key Steps:
    • Dehydration: Water is removed with ascending alcohol grades.
    • Clearing: Alcohol is replaced with xylene, making the tissue translucent.
    • Infiltration: Tissue is permeated with molten paraffin wax (at ~60°C).
    • Embedding: The infiltrated tissue is oriented in a mold and encased in a solid paraffin block.
    • Sectioning: The block is cut into extremely thin slices (4-6 µm) on a microtome.

⭐ Common artifacts include microtome knife marks (scratches from a dull blade) and "venetian blind" chatter (vibrations).

H&E Staining - The Classic Pink & Blue

  • The cornerstone of histological staining, providing fundamental morphological information. It's a differential stain based on charge.

  • Hematoxylin (H): A basic dye (net positive charge).

    • Stains acidic, basophilic structures blue/purple.
    • Examples: Nucleus (heterochromatin, nucleoli), ribosomes, rough ER.
    • 📌 Hematoxylin stains structures that are Heavy with nucleic acids blue.
  • Eosin (E): An acidic dye (net negative charge).

    • Stains basic, eosinophilic (or acidophilic) structures pink/red.
    • Examples: Cytoplasm, mitochondria, collagen, muscle filaments, RBCs.

H&E stain of fibrous tissue with basophilic nuclei

High-Yield Fact: Structures poor in charged molecules, like adipocytes (fat), glycogen, and the Golgi apparatus, do not stain well with H&E and appear clear or pale.

Special Stains - Beyond the Basics

  • Periodic Acid-Schiff (PAS): Stains glycogen, mucosubstances, and basement membranes magenta. Useful for identifying fungi and diagnosing glycogen storage diseases. PAS-positive, diastase-resistant material is typically mucin.

  • Prussian Blue: Detects ferric iron ($Fe^{3+}$), staining it blue. Crucial for identifying hemosiderin in conditions like hemochromatosis or pulmonary hemorrhage.

  • Congo Red: Specifically binds amyloid protein. The definitive test requires visualization under polarized light.

  • Silver Stains (e.g., GMS): Gomori Methenamine-Silver (GMS) is essential for visualizing fungi (e.g., Pneumocystis jirovecii) and basement membranes, staining them black.

  • Masson's Trichrome: Distinguishes collagen from muscle. Stains collagen/connective tissue blue-green and muscle/epithelium red/pink. Widely used to assess fibrosis.

⭐ With Congo Red stain, amyloid deposits exhibit a characteristic apple-green birefringence under polarized light-a pathognomonic finding.

High‑Yield Points - ⚡ Biggest Takeaways

  • Formalin fixation is the first and most critical step, preserving tissue by cross-linking proteins.
  • Tissue is dehydrated with alcohol, cleared with xylene, and infiltrated with paraffin wax for support.
  • Hematoxylin (H) is a basic dye that stains acidic, basophilic structures like the nucleus blue.
  • Eosin (E) is an acidic dye that stains basic, acidophilic structures like cytoplasm and collagen pink.
  • Remember: Basophilic = Blue (DNA, RNA); Acidophilic/Eosinophilic = Pink (proteins).
  • Be aware of processing artifacts, which can resemble pathology.

Practice Questions: Tissue preparation and staining

Test your understanding with these related questions

A 35-year-old woman presents to a pre-operative evaluation clinic prior to an elective cholecystectomy. She has a 5 pack-year smoking history. The anesthesiologist highly recommends to discontinue smoking for at least 8 weeks prior to the procedure for which she is compliant. What is the most likely histology of her upper respiratory tract's epithelial lining at the time of her surgery?

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Flashcards: Tissue preparation and staining

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The intermediate filament Desmin is found in _____ cells

TAP TO REVEAL ANSWER

The intermediate filament Desmin is found in _____ cells

muscle

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