3rd week 1 (tissue preparation) Flashcards
Tissue sources for histological studies:
(2)
- Animal models
- Post-mortem, pathology samples, surgical surplus
Pros in animal models (as tissue source)
(3)
possible to study different stages of disease (early to late)
effects of specific mutations
assesing therapeutic startegies
sample preparation
4 stages
fixing
processing
bedding
section
cons in animal models (2)
may not fully recapitulate human disease
ethical concerns
In what ways can human tissue be acquired for studies?
(3)
post-mortem donor
pathology samples
surgical surplus
pros (2) in human tissue studies compared to animal models
cons (3)
reduced need for animal research
better material for studying human disease
ethical concerns
limited supply
low availability of early stages of a disease
aims of tissue preparation (2)
methods (2, in general)
preserve as life-like as possible
prevent irreversible tissue/cell destruction (presence of pathogens)
Methods:
- chemical fixation
- cryopreservation
acetic acid, formaldehyde, ethanol, glutaraldehyde, methanol, picric acid
Are types of….?
Used for…?
types of fixatives
used for chemical fixation
Of chemical fixatives which one is:
- most used:
- gives best morphology & poor staining
- gives best staining & poor morphology
(acetic acid, methanol, glutaraldehyde, formaldehyde)
- most used: formaldehyde
- gives best morphology:
- gives best morphology & poor staining
glutaraldehyde
- gives best staining & poor morphology
acetic acid, methanol
(formaldehyde in the middle)
chemical fixatives stabilize …. and ….?
proteins and macromolecules (like carbohydrates)
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?
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.
Two types of chemical fixatives (different mechanism)
cross-linking fixatives
precipitating fixatives
ethanol, methanol are chemical fixatives and by working principle …. fixative (solid form).
Explain principle:
Precipitating
disrupts hydrophobic (water disliking) bonds between [aminoacids] and proteins. Causes them to irreversibly precipitate.
Two ways of fixating fresh tissue.
(2 different ways as how does the fixing fluid get into the tissue)
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
Factors affecting fixation quality? (4)
Rate of formalin penetrating tissue X mm / hour?
- 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
