Practical Side of Histology

Question 1

How are tissue slides prepared for a microscope?

Answer 1

1) FIXATION: blocks degradation of tissue
a) Chemical Fixation: neutral buffered formalin (NBF)
b) Freezing Fixation: cryostat is used, -20/30 for few mins (steps 2 & 3 not needed)

2) DEHYDRATION: remove water and replace with alcohol > lipids dissolve
3) INCLUSION: hardening the sample with hot paraffin/wax
4) Cutting: few microns thick to allow light through
5) Mounting then rehydration
6) STAINING: most commonly H&E

Question 2

Name and Describe the Types of Staining

Answer 2

1) Hematoxylin and Eosin (95% common)
- Hematoxylin: basic dye > binds acidic components (nucleic acids in ribosomes, DNA and RNA) > blue/purple
- Eosin: acidic dye > binds basic components (proteins, membranes, cytoplasm) > red
NB: structures not readily stained by H or E are called neutrophilic structures e.g. mucus

2) Trichrome: used to differentiate intercellular structures e.g. connective tissue from muscular
- Masson’s Trichrome:
* Hematoxylin: dark blue > nucleus
* Fuchsin: red > muscle, keratin, cytoplasm
* Aniline blue: clear blue > mucin, collagen
- Azan-Mallory: distinguishes ECM and displays connective tissue collagen fibres
* Red: nuclei, erythrocytes, neurofibrils
* Pale red: cytoplasm
* Blue: collagen fibres
* Azure: mucin
* Orange: muscular fibres

3) Prussian Blue/ Iron Hematoxylin
- Highlights tissues/cells containing iron
- Muscle and red blood cells

4) Periodic Acid-Schiff (PAS) > proteoglycans
- also for polysaccharides (glycogen)
- highlights surface of intestinal cells

5) Neurons
- Nissl > rER of neurons
- Silver and Gold > fibres & cytoskeletal elements
- Osmic acid > myelin
- Cresyl violet > proteoglycans

7) Verhoff Stain > stains elastin black

8) Romanovsky Stain > blood cells
- useful for highlighting types of WBC granules
* Pink: erythrocytes, eosiniphilic granules
* Purple: leukocyte nuclei, basophilic granules
* Blue: cytoplasm, monocytes and lymphocytes

9) Osmium Acid Stain > lipids black
- Lipids droplets and membranous structures
- Myelin sheath black

10) Metachromasia: ability to change colour in presence of particular substances
e. g. Granules in cytoplasm of mast cells are purple when stained by Toluidine Blue (a blue dye)

11) Oil Red O > lipids red/orange in unfixed frozen

Question 3

What are the different types of Artifacts?

Answer 3

PRE-HISTOLOGY

• ink from tattoos
• freckles (melanin) in skin samples

POST-HISTOLOGY - in tissue processing

• shrinkage caused by substances of different strengths > results in white stripes on sample
• bad cutting

Question 4

What is Immunohistochemistry?

Answer 4

Involves selective identification of ANTIGENS, expressed by the cells of a tissue section, by using ANTIBODIES able to specifically bind them

It is semi-quantitive:
- tells us if tissue is +/-ve for an antigen
- gives quantitative info through intensity of colour
> tumours are catagorised based on antigen expression

Question 5

Describe the two ways in which we can visualise the antigen-antibody interaction in IHC

Answer 5

1) Chromogenic IHC
- antibody is conjugated to an enzyme (peroxidase)
- enzyme can catalyse a colour-producing reaction

2) Immunofluorescence
- antibody is tagged to a fluorophore (fluorescien)

Question 6

Talk about Electron Microscopes

Answer 6

They use accelerated electrons as the source of illumination

1) TEM (transmission electron microscope)
Shows different densities of structures

2) SEM (scanning): shows surface of structures

Question 7

What are the two main characteristics of microscopes?

Answer 7

1) Magnification

2) Resolution Power