histological techniques and tissue identification

• Basophilia


  Tissue structures with an affinity for Haematoxylin (and other basic dyes)
• Acidophilia


  Tissue structures with an affinity for acid dyes
• Eosinophilia


  Tissue affinity for Eosin (dye term)
• Rate dependent staining processes

  1. Dye binds progressively to tissue structures, staining them more intensely the longer the tissue remains in the dye bath

  2. Dye is selectively lost or removed from a tissue as a means of distinguishing or differentiating between components with stronger or weaker affinities for the dye (regressive staining)
• Haematoxylin


  Natural product extracted from Haematoxylum campechianum (logwood) in Mexico, requires oxidation to haematein before use as a stain, stains basophilic structures like nucleic acids and ribosomes
• Haemalum


  Harris's haematoxylin uses aluminium salts as a mordant, requires rinsing in tap water to blue the section, used regressively with controlled differentiation in acid alcohol followed by rebluing in tap water
• Eosin


  Variety of related acidic dyes that bind to basic (Eosinophilic) compounds, derived from fluorescein, provides red cytoplasmic stain
• After this session you should be able to: Identify key structures in tissue samples A, B and C, Carry out H&E staining of tissue sections
• Tissue section A and B are already stained, you only need to stain C
• Haematoxylin & Eosin staining Protocol

  1. Dewax section in Histoclear

  2. Absolute alcohol

  3. 90% alcohol

  4. 70% alcohol

  5. Wash in distilled water

  6. Immerse in Harris's haematoxylin

  7. Wash in distilled water

  8. Differentiate in 1% Acid Alcohol

  9. Wash in tap water

  10. Immerse in Scott's solution

  11. Wash in running tap water

  12. Wash in distilled water

  13. Immerse in Eosin

  14. Dehydrate in 70% alcohol

  15. Dehydrate in 90% alcohol

  16. Dehydrate in absolute alcohol

  17. Clear in Histoclear
• Mounting slides


  1. Drain excess Histoclear from the slide

  2. Wipe excess Histoclear from the back of the slide

  3. Place the slide on a level surface, and apply a drop of DPX

  4. Hold the cover slip at a 45° angle to the surface of the slide, and allow the bottom edge to touch the drop

  5. When the drop has spread along the edge of the slip gently lower the cover slip and allow the mounting media to spread slowly

  6. Excess mounting medium may be removed while wet with a tissue

  7. Leave in the fume hood to dry
• Please work on trays at all times since the spillage of these dyes on the benches is extremely difficult to remove
• Whilst waiting for slide C to dry, examine the pre-stained slides A and B
• Tissue A - Liver

  • HEPATIC LOBULE (bile canaliculi, sinusoidal capillaries, hepatocytes)
  • HEPATIC VEIN
  • PORTAL TRIAD (artery, vein, bile duct)
• Tissue B - Skin

  • Epidermis
  • Dermis
  • STRATUM CORNEUM (cornified or horny layer)
  • PRICKLE CELL LAYER
  • DERMIS
  • FAT
  • SWEAT GLAND
  • HAIR FOLLICLE
  • BLOOD VESSELS
  • SEBACEOUS (oil) GLAND
  • LYMPH VESSEL
  • SENSORY NERVE RECEPTOR
  • HAIR
  • PORES
• Tissue C - Duodenum


  • EPTHELIAL LAYER OF CELLS
  • VILLUS
  • LAMINA PROPRIA
  • MUSCULARIS MUCOSAE
  • INTESTINAL CRYPTS (crypt of Lieberkuhn)
  • BRUNNER'S GLAND
  • SEROSA
• Submucosa


  Brunner's glands secrete alkaline mucus (neutralises gastric acid in chyme)
• Muscularis externa


  Segmentation contractions (mix chyme in small intestine) and weak peristalitic contractions (move chyme along small intestine)
• The muscle in the muscularis externa is arranged with an outer thinner layer that is longitudinal and an inner thicker layer that is circular
• The absorptive epithelium proper has one layer of cells
• There are two cell types in the epithelium
• Cell types in the epithelium

  • Rectangular, elongated nuclei (enterocytes/absorptive cells)
  • Cup shaped (goblet cells, do not stain)
• The ratio of the two cell types is approximately 10:1 (enterocytes:goblet cells)
• Enterocytes


  Absorptive cells
• Goblet cells


  Cup shaped cells
• Cells in the process of dividing (mitosis) can be seen at the base of the intestinal crypts/crypts of Lieberkuhn