Tissue staining

Question 1

What is the purpose of staining tissues?

Answer 1

Thin tissue slices are colourless.Staining (with chemical dyes or heavy metals) helps to reveal detail in these samples

Question 2

What is another means of revealing detail in colourless samples, besides staining with chemical dyes or heavy metals?

Answer 2

Manipulate optical pathways on a light microscope (phase contrast, darkfield, differential interference contrast)

Question 3

Why is fresh tissue a problem, and how is this problem overcome?

Answer 3

Fresh tissue is soft, decays quickly (autolysis) and cannot be sliced thinly enough to view under a microscope.Solution = tissue fixation

Question 4

What are the steps in tissue preparation?

Answer 4

• Fixation
• Processing
• Sectioning
• Mounting
• Staining

Question 5

Explain the steps of tissue preparation

Answer 5

Fixation: preserves tissue morphology and prevents autolysisProcessing: dehydration, clearing, infiltration, embeddingSectioning: thin sections cut on a microtome or cryomicrotomeMounting: sections mounted onto microscope slideStaining: chemical dyes impart colour contrast

Question 6

What is the purpose of tissue fixation?

Answer 6

Preserves tissue morphology and prevents autolysis

Question 7

What are the types of tissue fixation?

Answer 7

Chemical and physical

Question 8

Explain two types of chemical fixatives

Answer 8

Cross-link fixatives:aldehydes and oxidising agents – form cross-links with their targets, stabilizing tissue structure
Coagulants: EtOH, MeOH,acetic acid – coagulate/precipitate proteins. Conformation changes by disrupting hydrophobic interactions. Rarely used

Question 9

Explain a type of physical fixation

Answer 9

Physical fixation:

Freeze in liquid N2 with cryo-protectant (isopentane or propane)

Question 10

Name two types of cryo-protectants used in physical fixation

Answer 10

isopentane and propane

Question 11

What are the 4 steps involved in processing?

Answer 11

• Dehydration
• Clearing
• Infiltration
• Embedding

Question 12

What occurs during dehydration?

Answer 12

Sample placed in ethanol to remove water

Question 13

What occurs during clearing?

Answer 13

xylene, toluene and chloroform remove the ethanol

Question 14

What occurs during infiltration?

Answer 14

xylene is replaced with molten paraffin which infiltrates tissue and provides a hard matrix for cutting

Question 15

What occurs during embedding?

Answer 15

Tissue is placed in a mould containing molten wax/resin. Wax/resin then hardens
Ensure tissue is orientated into the plane of preference

Question 16

What is an important consideration during embedding?

Answer 16

Ensure tissue is orientated into the plane of preference

Question 17

What occurs during sectioning?

Answer 17

Thin 5 um sections of wax-hardened tissue are cut on a microtome

Question 18

On what device is wax-hardened tissue cut?

Answer 18

Microtome

Question 19

To what thickness are wax-hardened tissues cut?

Answer 19

5um

Question 20

What occurs during cryosectioning?

Answer 20

Tissue is embedded in support medium OCT (optimal cutting temperature), then frozen. Sections are cut on a cryostat/cryomicrotome
(can either use prior chemically fixed tissue and freeze it, or use unfixed frozen tissue)

Question 21

On what device are cryo-sectioned samples cut?

Answer 21

Cryostat/cryotome

Question 22

What occurs during mounting?

Answer 22

Tissue slices are placed in a warm water bath before being mount onto microscope slide

Question 23

What occurs during staining?

Answer 23

Chemical dyes impart colour contrast. Different dyes are used to stain different cellular components.

Question 24

What are dyes composed of?

Answer 24

A benzene molecule forms the backbone of dyes, to which a chromophore is added. (an organic molecule component which absorbs light)

Question 25

Name 4 different mechanism of dye-staining

Answer 25

Acid-base interactions Permeability and displacementDeposition/impregnation/precipitationHistochemistry 

Question 26

Provide an example of an acid-base dye stain, and explain the principles underlying it

Answer 26

H&E stain basic (+) dye (haematoxylin) binds to acidic (-) components e.g., DNA and RNA acidic (-) dye (eosin) binds to basic (+) components e.g., cytoplasmic proteins

Question 27

What does H&E stand for?

Answer 27

H=haematoxylin | E=eosin

Question 28

What are 3 problems with H&E staining, and what are solutions to these problems?

Answer 28

Problems * Can’t distinguish between different tissue components with similar charge – all display same colour * Fine extracellular fibres not visible. Cytoplasm and extracellular fibres show similar staining * Mucopolysaccharides and lipids are unstainedSolutions * Use trichrome stains * Use metal salts/deposition * use periodic acid-Schiff stain (PAS) 

Question 29

Provide an example of a permeability and displacement stain, and explain the principles underlying it

Answer 29

Trichrome stain Trichrome stains are acid dyes (i.e., stain basic components) with the same net charge. They stain according to relative molecular weight and use principles of permeability and displacement The permeability of the dye depends on its relative molecular weight, as well as that of the tissue components: -lower molecular weight dyes/diffusible dyes stain penetrate easily and stain all tissue components, including denser, compact structures -higher molecular weight dyes/colloid dyes penetrate more permeable tissue structures such as collagen Larger dye molecules displace smaller dye molecules from permeable structures

Question 30

What does the permeability of trichrome dyes depend on?

Answer 30

* Relative molecular weight of dye | * Relative molecular weight of tissue components

Question 31

What does the permeability of trichrome dyes depend on?

Answer 31

* Relative molecular weight of dye | * Relative molecular weight of tissue components

Question 32

What is another name for low molecular weight dyes, and how do they work?

Answer 32

diffusible dyes  | penetrate easily and stain all tissue components, including denser, compact structures

Question 33

What is another name for high molecular weight dyes, and how do they work?

Answer 33

colloid dyes  penetrate more permeable tissue structures such as collagen larger dye molecules displace smaller dye molecules from permeable structures

Question 34

Provide an example of a deposition/impregnation/precipitation dye and explain the principles underlying it

Answer 34

Metal salt stain e.g. reticulin stain These bulk up fine structures and make them visible The process is as follows: • Tissue is treated with oxidising agent which has an affinity for silver, to enhance subsequent staining.  • Tissue is then sensitized using an iron alum solution which targets and binds to the tissue element (retic fibres) • Silver solution impregnates the fibres by replacing the sensitizer with metal salts • Reducing agent is added, causing metal salts to be reduced to visible metallic form at binding site  

Question 35

Provide an example of a histochemistry dye and explain the principles underlying it

Answer 35

Enzyme histochemistry Chemical reaction between tissue and colourless staining solution to form a colour compound Can be used to visualise enzyme activity in a tissue. Cannot use chemical fixation, as this may inactivate the enzymes.  Frozen sections are incubated in solutions containing substrates for the enzyme of interest.  Active enzymes react with the substrate to form a primary reaction product which is colourless. The enzyme cleaves hydrogen from the substrate, which then which then combines with colourless tetrazolium dye salt to form an insoluble purple-blue precipitate (formazan) marking the site of enzyme activity.  

Question 36

Why can't chemical fixation be used in enzyme histochemistry?

Answer 36

May inactivate the enzymes

Question 37

What is the name of a colourless dye salt used in enzyme histochemistry?

Answer 37

Tetrazolium dye salt

Question 38

What is the name of an insoluble purple-blue precipitate used in enzyme histochemistry?

Answer 38

formazan

Question 39

Explain the principle of the periodic acid-Schiff stain (PAS)

Answer 39

PAS reacts chemically with tissue, forming a deep magenta colour in the presence of mucopolysaccharides (not lipids).