3rd week 1 (tissue preparation)

Question 1

Tissue sources for histological studies:

(2)

Answer 1

• Animal models
• Post-mortem, pathology samples, surgical surplus

Question 2

Pros in animal models (as tissue source)

(3)

Answer 2

possible to study different stages of disease (early to late)

effects of specific mutations

assesing therapeutic startegies

Question 3

sample preparation

4 stages

Answer 3

fixing

processing

bedding

section

Question 4

cons in animal models (2)

Answer 4

may not fully recapitulate human disease

ethical concerns

Question 5

In what ways can human tissue be acquired for studies?

(3)

Answer 5

post-mortem donor

pathology samples

surgical surplus

Question 6

pros (2) in human tissue studies compared to animal models

cons (3)

Answer 6

reduced need for animal research

better material for studying human disease

ethical concerns

limited supply

low availability of early stages of a disease

Question 7

aims of tissue preparation (2)

methods (2, in general)

Answer 7

preserve as life-like as possible

prevent irreversible tissue/cell destruction (presence of pathogens)

Methods:

• chemical fixation
• cryopreservation

Question 8

acetic acid, formaldehyde, ethanol, glutaraldehyde, methanol, picric acid

Are types of….?

Used for…?

Answer 8

types of fixatives

used for chemical fixation

Question 9

Of chemical fixatives which one is:

• most used:
• gives best morphology & poor staining
• gives best staining & poor morphology

(acetic acid, methanol, glutaraldehyde, formaldehyde)

Answer 9

• most used: formaldehyde
• gives best morphology:
• gives best morphology & poor staining

glutaraldehyde

• gives best staining & poor morphology

acetic acid, methanol

(formaldehyde in the middle)

Question 10

chemical fixatives stabilize …. and ….?

Answer 10

proteins and macromolecules (like carbohydrates)

Question 11

formaldehyde, glutaraldehyde and all -aldehydes are:

… linking fixatives.

Formaldehyde and glutaraldehyde create …. bonds between proteins in the tissue

All -aldehydes anchor proteins (vrt. ed. lause) and therefore have bad/good morphology?

Maintains protein tertiatory (3D) structure, fixing process is fast/slow?

Answer 11

cross-linking fixatives

Formaldehyde and glutaraldehyde create covalent (vrt. atoms) bonds between proteins in the tissue

All -aldehydes anchor proteins and therefore have good morphology.

Maintains protein tertiatory (3D) structure, but fixing process is slow.

Question 12

Two types of chemical fixatives (different mechanism)

Answer 12

cross-linking fixatives

precipitating fixatives

Question 13

ethanol, methanol are chemical fixatives and by working principle …. fixative (solid form).

Explain principle:

Answer 13

Precipitating

disrupts hydrophobic (water disliking) bonds between [aminoacids] and proteins. Causes them to irreversibly precipitate.

Question 14

Two ways of fixating fresh tissue.

(2 different ways as how does the fixing fluid get into the tissue)

Answer 14

Immersion fixation

(tissue placed in fluid0 [glass jar and stirred gently]

Perfusion fixation

Fixing fluid injected into the circulatory system (replaces blood)

Gets quickly into the organ

Gives superior preservation (preferred method)

https://www.usf.edu/research-innovation/comparative-medicine/documents/training/perfusion.pdf

Question 15

Factors affecting fixation quality? (4)

Rate of formalin penetrating tissue X mm / hour?

Answer 15

• changes in pH
• length of incubation time in fixative
• size of specimen (piece of tissue) usually dissected to less than 5 mm3
• temperature (4’C [cool] slows degerative changes but also fixation penetration rate)

Formalin penetrates tissue 1mm/hour

Question 16

Cryopreservation

= freezing tissue ….. without fixation (or first fix then freeze)

Answer 16

rapidly

Question 17

Snap freezing is done with (2)

Answer 17

dry ice (cooled and condensed carbon dioxide, CO2) or liquid nitrogen (LN2)

Question 18

Pros of cryopreservation

• fast / slow
• minimal changes to ….. ….
• rapid cooling, - X degrees Celcius
• rapid cooling minimises damage to the tissue, why?

Answer 18

• fastest method
• protein structure
• minus 70 degrees Celcius
• ice-crystal artefact, water doesn’t crystalise when cooled fast

Question 19

Cons of cryopreservation:

• Morphology is good / poor
• Tissues stored at -X ‘C, never the less …. still occurs
• Storing requires…

Answer 19

• poor
• minus 80’C, degradation over time
• special cold storage (-80!)

Question 20

Embedding purpose

• Sample embedded in a ….. medium
• gives …. for the tissue structure
• sufficient rigidity to enable ….

Answer 20

• solid medium
• support
• cutting of thin sections

Question 21

Embedding means:

Plastic resins

Paraffin wax

Gelatine and Agarose

soft media&thick section / hard media&thin section / most common

Question 22

aims of preparation and embedding (generally)

Preparation prevents …

Embedding prevents tissue … when cutting

Answer 22

degeneration

distortion

Question 23

difference between OCT and paraffine wax?

Answer 23

commercial, water-soluble embedding material,

paraffine wax not water soluable

Question 24

Steps of embedding:

order and explain

infiltrating / dehydrating / cleaning / embedding

Answer 24

dehydrating / cleaning / infiltrating / embedding

• dehydrating all the water out using alcohol (70%, 90%, 100%)
• cleaning alcohol away by a solvent that mixes with both; alcohol and paraffin wax (xylenes, Histo-clear)
• infiltrating the tissue with molten paraffin wax (replacing xylene/cleaning aid)
• embedding orientating in a metal mould, fill with molten paraffin wax, allow to cool 4 ‘C, followed by microtomy or storing