Histology

Question 1

Define histology?

Answer 1

The study of cellular structure and function of the body

Question 2

Define tissue structure, cellular structure and sub-cellular structure?

Answer 2

Tissue structure: how cells combine together with extracellular material and each other to form a tissue
Cellular structure: cell shape? and how the components inside cells are organized to support that cells specific function
Sub-cellular structure: analysis of organelles and inclusions

Question 3

What are the 4 different tissue types

Answer 3

Epithelial tissue
Connective tissue
Muscular tissue
Nervous Tissue

Question 4

What does the ECM consist of and what is its function

Answer 4

ECM consists of many molecules, which are highly organized and form complex structures like collagen fibrils and basement membranes.
Its function is to:
- Transport Nutrients and ions
- Mechanical support for cells
- Carry away metabolites and secretory waste.

Question 5

What are the stages of tissue preparation?

Answer 5

• Fixation
• Dehydration
• Clearing
• Embedding
• Sectioning
• Staining

Question 6

What is the aim of tissue fixation?

Answer 6

To allow tissues to be in their closest living state in which they can be observed and prevent autolysis.

Question 7

Why is dehydration done with an alcohol degrading series

Answer 7

To prevent the distortion of cells via diffusion currents

Question 8

What factors does the clearing agent depend on?

Answer 8

• The type of tissues to be processed, and the type of processing to be undertaken.
• The processor system to be used.
• Intended processing conditions such as temperature, vacuum and pressure.
• Safety factors
• Cost and convenience
• Speedy removal of dehydrating agent
• Ease of removal by molten paraffin wax
• Minimal tissue damage

Question 9

What histological purposes is paraffin wax adapted for?

Answer 9

Decreased melting point, increased hardness and increase adhesion to sample and embedding material and improved ribboning.

Question 10

What steps must be taken if the stain is not compatible with the paraffin embedding material.

Answer 10

• Slides cleared in Xylene
• Rehydrated through alcohol series
• Washed in water to remove alcohol
• Stained
• Dehydrated
• Cleared in Xylene
• Mounted using xylene medium and glass coverslip

Question 11

What does the H&E stain do?

Answer 11

H & E is a charge-based, general purpose stain. Haematoxylin: cationic dye (+ charge) that binds to negatively charged (acidic) structures in the cell. Nucleus = Blue
Eosin: anionic dye (- charge) adheres to basic structures in the cell. Amine groups (NH3+) on proteins make cytoplasm = Pink

Question 12

What does the DAPI stain do?

Answer 12

Fluorescent stain which binds to A-T rich regions (minor grove) in DNA. DAPI can pass through an intact cell membrane, it can be used to stain both live and fixed cells. Double stranded DNA, therefore, nuclei- Blue

Question 13

What does the Alcian stain do?

Answer 13

Alcian blue is a mucin stain that stains certain types of mucin blue. Cartilage is also stained blue. It can be used with H&E, nuclear fast red, and with van Gieson stains.
Acid mucins- Blue
Proteoglycans- Blue
Nuclei- Red/Black

Question 14

What does the OIL RED O stain do?

Answer 14

Oil Red O is used to stain fat in unfixed frozen sections. Processing may remove the fact content from cells and tissues
Fat- Brilliant red
Nuclei- Blue

Question 15

What does the Miller Sirius stain do?

Answer 15

Combination stain viewed under Köhler illumination and phase contrast
Köhler
Elastin - Dark purple/ Black
Collagen- Red/ Pink
Phase contrast 
Collagen fibres are birefringent

Question 16

What does Masson trichrome stain do?

Answer 16

Nuclei and other basophilic (basic liking) structures= blue
cytoplasm, muscle, erythrocytes and keratin = bright red
Collagen is stained= green or blue
Depending on which variant of the technique is used

Question 17

What is reticulin stain for?

Answer 17

Reduction of silver ions into silver metal and its deposition on the reticulin fibers (mainly collagen type III).
Reticulin fibers- Black
Collagen fibers- Brown
Nuclei- Pink

Question 18

What methods can be used for antigen retrieval?

Answer 18

Enzymatic digestion
Citric Acid
EDTA
Heat

Question 19

What are the advantages and disadvantages of immunohistochemistry?

Answer 19

Advantages
High specificity for molecular species
Can be used for light, confocal, or electron microscopy
Disadvantages.
Time consuming & expensive
Fixation can interfere with Ab binding
Reproducibility - false positives - cross reactivity
Difficult to get Abs to small molecules
Qualitative

Question 20

What is microscopy phase contrast?

Answer 20

Allows viewing of unstained specimens

Question 21

Define DIC Microscopy?

Answer 21

Allows real 3d images real time cell images via polarized light.

Question 22

Define Fluorescence Microscope?

Answer 22

Uses specific light frequencies to excite a fluorophore at specific sites.

Question 23

What is a confocal microscope?

Answer 23

A laser is positioned at specific points at the specimen and creates a 3d image.

Question 24

What are the advantages and disadvantages of the confocal microscope?

Answer 24

Advantages
Excellent resolution in thick samples
Greater number of fluorophores as specific wavelengths used to illuminate samples. Collects light from a single focal plane
Disadvantages
Photo bleaching and phototoxicity
Increased sensitivity to noise
Technical method - labour intensive

Question 25

What are the three different types of ELISA test and define this?

Answer 25

Sandwich ELISA- antibody coated well, wash, add antigen to be measured, add conjugated enzyme secondary antibody. Then add substrate to measure colour.
Indirect ELISA- Antigen coated well, wash, add specific antibody, wash, add enzymes conjugates secondary antibody, wash, add substrate and measure colour.
Competitive ELISA- Incubate antibody with the antigen to be measured then add the mixture to antigen coated well, wash, add enzyme conjugated secondary antibody, wash add substrate and measure colour.