Principles of Histology Staining Flashcards
What other chemical bonds are involved in selectivity and exceptional circumstances, in relation to staining?
- Hydrogen bonds: important in the staining of amyloid by Congo red
- Van der Waals: important in keeping tissue fixed to the slides
- Covalent bonding: important in attaching dyes to antibodies in immunofluorescence
- Hydrophobic bonds: important in selectivity and play a major role in the staining of lipids
What are mordants?
Metal salts that help bind dyes to tissues.
What is a dye lake?
The dye and Mordant complex = dye lake
What is the effect of a mordant?
Gives greater stability to the stain so that it is less easily removed by water, alcohol or weak acid
Give some examples of mordants.
Haemotoxylin and aluminium potassium/ammonium sulphate
What are the 2 types of staining?
1) progressive staining
2) Regressive staining
What is progressive staining?
The dye is applied gradually to the section until the desired density of colour is reached
What is regressive staining?
More common to use mordanted stains regressively. Acids or excess mordant can be used to differentiate the tissue.
What is metachromasia?
Some dyes exhibit this behaviour which means they can stain different tissue components a different colour.
What is haemotoxylin?
The most commonly used stain in any routine histopathology department. It is used as a nuclear stain which depicts the chromatin pattern in a dark blue/black colour.
What cellular structures does the Periodic Acid-Schiff reaction show?
Demonstrates various cellular structures including carbohydrates, mucins, basement membranes and viable fungi. Histochemical method.
What are 2-step chemical reactions involved in the PAS Reaction?
1) Periodic Acid
-oxidises sugars to form aldehyde groups
periodic acid is chosen as it stops the oxidation process once these aldehyde groups are formed
2) Schiff’s reagent
- contains fuchsin that has been treated to displace the chromophore group resulting in loss of colour
- reaction with aldehyde groups restores the chromophore to give a strong red/purple colour
What causes false positive PAS reactions?
- improper schiff reagent preparation. dye may not have fully decolourised, may have deteriorated, out of date etc.
- certain fixatives can cause artificial production of aldehyde groups within the tissue
- preformed aldehydes can also occur naturally in small amounts in tissues
What causes false negative PAS reactions?
-usually associated with poor technique, timing or reagent preparation
What does the Perls Prussian Blue stain demonstrate?
- demonstrates the presence of iron within the tissue
- hemosiderosis: excess iron i the liver
What does the Oil Red O stain demonstrate?
- demonstrates the presence of lipid within the tissue
- this technique can only be performed on frozen sections
What does the Orcein stain demonstrate?
- presence of hepatitis B surface antigen.
- positive cells have “ground glass” like appearance.
What is Silver Staining used for?
AKA silver impregnation
- involves the reduction of silver ions in solution to leave a very fine deposition of silver and silver oxide
- structures such as reticulin actually become plated with silver rather than being reversibly bound like other tinctorial dyes.
What is Reticulin Silver staining?
- Oxidisation: first hexose groups are oxidised to form aldehydes
- sensitisation: aldehydes reduce silver nitrate solution in solution to leave a deposition of silver ions
- developing: transferring the section into formaldehyde further reduces the diamine silver
- toning: silver is replaced with metallic gold and the colour of the reticulin fibres change from brown to black
What does the reticulin stain demonstrate?
-the structure or pattern of reticulin fibres based upon their argyrophilic nature
What does Van Gieson’s stain demonstrate?
(mixture of picric acid and acid fuchsin)
-differentiates collagen from other connective tissues
When cellular components are stained with Van Gieson’s stain, what results are observed?
Nuclei - Blue
Collagen - Bright Red
Cytoplasm, Fibrin, Muscle, RBCs - Yellow
What are the functions of the epithelium?
- protection
- absorption
- filtration
- excretion
- secretion
What features are characteristic of the epithelium?
- cells are in close proximity to each other and form sheets
- epithelial cells have an apical and basal surface
- basal surface rests of the membrane
- avascular
- regenerative capacity (undergo mitosis)
