Anatomy L3 - Introduction to Histology
Terms in this set (10)
Steps of tissue processing
1. Obtaining a fresh specimen
5. Wax infiltration
6. Embedding or blocking out
- The specimen is placed in a liquid fixing agent (fixative) such as formaldehyde solution (formalin).
-This will slowly penetrate the tissue causing chemical and physical changes that will harden and preserve the tissue and protect it against subsequent processing steps.
- Most of the water in a specimen must be removed before it can be infiltrated with wax.
-This process is commonly carried out by immersing specimens in a series of ethanol (alcohol) solutions of increasing concentration until pure, water-free alcohol is reached.
- Because wax and ethanol are largely immiscible. We therefore have to use an intermediate solvent that is fully miscible with both ethanol and paraffin wax.
- This solvent will displace the ethanol in the tissue, then this in turn will be displaced by molten paraffin wax.
- This stage in the process is called "clearing" and the reagent used is called a "clearing agent".
- The tissue can now be infiltrated with a suitable histological
wax. typical wax is liquid at 60°C and can be infiltrated into tissue at this temperature then allowed to cool to 20°C where it solidifies to a consistency that allows sections to be consistently cut.
Embedding or blocking out
- Now that the specimen is thoroughly infiltrated with wax it
must be formed into a "block" which can be clamped into a microtome for section cutting.
Common stains used to study tissues
• Acidic stains
• H&E stains
• Basic stains
• Neutral stains
• H&E contains the two dyes haemotoxylin and eosin.
• Eosin is an acidic dye: It stains acidic (or acidophilic) structures red or pink.
• Haematoxylin can be considered as a basic dye used to stain basic (or basophilic) structures a purplish blue. Thus the nucleus is stained purple.
Basophilic (purple) structures
- DNA (Heterochromatin and nucleolus) in the nucleus
- RNA in ribosomes and rough ER
- Extracellular materials (i.e carbohydrates in cartilage)
Acidophilic (pink) structures
- Most proteins in cytoplasm
- Cytoplasmic filaments in muscle cells
- Intra-cellular membranes
- Extracellular fibres