Preparation of samples for histological examination by light and electron microscopy
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Sampling[edit | edit source]
- 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[edit | edit source]
- 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[edit | edit source]
- 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[edit | edit source]
- 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[edit | edit source]
- 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[edit | edit source]
Mescher, A. and Junqueira, L., 2018. Junqueira's basic histology. New York: McGraw-Hill, pp. 1-4.
