Preparation of samples for histological examination by light and electron microscopy

Sampling

  • Purpose: obtain specimen of cells or tissue
  • Size: 0.5-1 cm3 for light microscopy, ~1 mm3 for electron microscopy
  • Methods: biopsy (excision, puncture, curettage, ascites, and aspiration) or necropsy

Fixation

  • Purpose: stabilize cell and tissue structures by denaturing them. This is necessary, since freshly removed tissues are
    • chemically unstable
    • will dry up and shrink
    • suffer from hypoxia and bacteria
    • will autolyse (degrade via own enzymes)
  • A good fixative must
    • preserve the structure well
    • quickly penetrate into the tissue block
    • not interact negatively with the staining
  • Most commonly used substances include Formaldehyde for LM (12-24h); Glutaraldehyde for EM (1-3h).
  • Ethanol, organic acids, inorganic acids, heavy metal salts, or compounds are also used
  • Excess fixative is rinsed off with water
  • Water is removed via ascending series of ethanol
  • To allow embedding medium to enter, ethanol is cleared with a solvent miscible in both (ex: xylen)

Embedding

  • Purpose: give firm texture to the sample to allow thin cutting
  • Hard tissues (like dental or bone) require softening by acid (decalcification), or grinding to thin specimens
  • Infiltration: tissue is placed into molten embedding medium (typically paraffin for LM, epoxide for EM)
  • Hardening at room temperature
  • Stable method: Formalin-Fixed-Paraffin-Embedded (FFPE). Many applications.

Cutting

  • Microtomes are used to precisely control thickness
  • Types: sliding, rotary, cryotomes, ultramicrotomes
  • Cryostat: rotary microtome in freezing box; used to cut frozen tissue without embedding
  • Thickness: 5-10 μm (light microscopy), 70-100 nm (electron microscopy)

Staining

  • Before starting, one must affix sections to microscopic glass using albumin or gelatin
  • Purpose: cells and their contents are usually colorless and thus invisible without staining for light microscopy. Electron microscopy uses heavy metals instead of staining for visualization
  • Chromophilic compounds have high affinity for dyes, chormophobic ones do not.
  • Basophilic components take up basic dyes (ex: nucleic acids); acidophilic components take up acidic dyes (cytoplasm, ionized proteins). Eosinophilic compounds take up eosin (ex: collagen)
  • Routine staining visualizes all tissue components; special staining visualizes a particular structure.
  • Mounting: attach cover slip with transparent adhesive over stained sample on slide
Routine Stain cell nucleus cytoplasm collagen erythrocytes muscle fibers Examples
Hematoxylin-Eosin (HE) blue pink-red pink red red Muscle
Hematoxylin-Eosin-Saffron (HES) blue pink-red yellow red red Esophagus
Azocarmine G-Aniline blue-orange G (AZAN) red red blue orange red Umbilical cord
Special Stains Stained Structures
Gomori impregnation reticular and nerve fibers - black
Oil red lipids - red
Orcein, Resorcin, Fuchsin (elastic) elastic fibers - brown/red-brown
Periodic acid Schiff (PAS) Reaction polymers incorporating sugars - red
Pappenheim blood cells
Schmorl fine bone structures
Sudan black lipids - blue-black
Toluidin blue/alcian blue cartilage, bone, ECM - blue

References

Mescher, A. and Junqueira, L., 2018. Junqueira's basic histology. New York: McGraw-Hill, pp. 1-4.