Histology Notes: Preparation, Staining, and Evaluation

Objectives

  • Explain how histological sections are prepared and their utility.
  • Compare processing and sectioning of frozen vs. paraffin-embedded tissues.
  • Prepare a Hematoxylin and Eosin (H&E) stain of formalin-fixed paraffin-embedded mouse cardiac tissue.
  • Evaluate the quality of an H&E-stained tissue section.

Histology Overview

  • Definition: Science that studies cellular anatomy of tissues using a microscope.
  • Purpose: Essential tool in science and medicine (e.g., diagnosing diseases and classifying cancers).
  • Professionals: Histotechnologists/Histotechnicians (often referred to as “histotechs”).
  • Example: H&E stain of a liver biopsy showing metastatic cancer cells.

Preparing Tissues for Histology

  • Importance: Tissue decays rapidly after removal; fixation halts this process.
  • Fixation Process:
    • Fixatives are essential to preserve tissue structure.
    • Common fixatives include ethanol, acetic acid, acetone, and formaldehyde.
    • Mechanisms:
    • Precipitating Fixatives: E.g., alcohols that precipitate proteins and nucleic acids; causes artifacts (structures not in original tissue).
    • Crosslinking Agents: E.g., formaldehyde; forms covalent bonds to retain tissue structure with fewer artifacts.
    • Human and animal tissues are usually fixed in 10% neutral buffered formalin (pH-balanced solution with 4% formaldehyde).

Sectioning Tissues

  • Technique: Thin slices (3-8 μm) cut using a microtome;
  • Embedding Process:
    • Paraffin embedding involves dehydration with alcohols, xylene, and infiltration with melted paraffin.
    • Produces sturdy paraffin blocks to be sliced into thin sections.
    • Frozen sections use cryoembedding medium and rapid freezing.
    • Prepared using a cryostat (a refrigerated microtome).

Staining the Sections

  • Purpose: Improve visibility of transparent tissues under a microscope.
  • Common Stains:
    • Hematoxylin and Eosin (H&E): Most common for animal tissues.
    • Hematoxylin: Basic dye; stains DNA blue/purple (nuclei).
    • Eosin: Acidic dye; stains proteins pink (cytoplasm).
    • Special Stains:
    • Periodic Acid Schiff: Binds glycogen; helps identify abnormal glandular tissue.
    • Masson’s Trichrome: Highlights collagen deposits (e.g., in scar tissue).

Staining Procedure (H&E)

  • Day 1: Preparation and staining of mouse heart tissue.
    1. Examine Slides: Note any artifacts; label properly.
    2. Staining Steps: Follow a series of solutions to deparaffinize, dehydrate, and stain.
    • Key Steps:
      • Citrisolv (deparaffinization).
      • Harris’ Hematoxylin (1 min).
      • Eosin (45 sec).
      • Mount coverslip with mounting medium after staining.

Evaluation of Stained Slides (Day 2)

  • Examine under 100x and 400x magnification.
  • Identify features:
    • Locate cardiomyocytes, endothelial cells, and fibroblasts.
    • Assess stain quality:
    • Nuclei should be blue/purple; cell cytoplasm light pink.
    • Detect artifacts (e.g., wrinkles, tears, spaces).
  • Comparative Analysis: Compare with commercial slides for differences.

Cleanup Procedures

  • Dispose of gloves properly and return lab coats and goggles to storage.
  • Ensure work areas are cleaned with 70% ethanol.