Immunohistochemistry: sample preparation Flashcards
Why examine tissues and cells?
To study tissue anatomy and cytoarchitecture
Distribution of proteins
Pathological changes in disease
Use of histological study of tissues
In clinical diagnostics of neuropathology
In basic and translational research
Name tissue sources
Animal models
Post-mortem
Pathology samples
Surgical material
Advantages of animal models
Study different stages of a disease
Study effects of specific mutations
Can assess therapeutic strategies
Disadvantages of animal models
Nor gully recapitulate human disease
Ethical concerns
Advantages of post-mortem donor tissue, tissue samples, and surgical surplus
Reduce the need for animal research
Arguably better for studying human disease
Disadvantages of post-mortem donor tissue, tissue samples, and surgical surplus
Ethical concerns
Limited tissue supply
Low availability of early stages of the disease
Which process starts after the removal of live tissue?
Irreversible apoptosis and lysis
Which tissues are more prone to cellular damage?
Post-mortem tissues
Aims of tissue preparation
Preserve tissue in a life-like manner as possible
Prevent irreversible cell/tissue destruction
Methods of tissue preparation
Chemical fixation and Cryopreparation
Type of chemical fixative which preserves morphology better but staining is poor
Glutaraldehyde
Type of chemical fixative which has intermediate preservation of morphology and staining
Formaldehyde
Types of chemical fixatives in which staining is better but has poor morphology preservation
Acetic acid and methanol
Use of chemical fixatives
Stabilize proteins and other macromolecules
Characteristics of glutaraldehyde and formaldehyde
Fixation process slow Cross-linking fixatives Anchor proteins Create covalent bonds between proteins in tissue Preserve protein tertiary structure
Characteristics of ethanol and ethanol
Disrupt hydrophobic bonds between proteins
Irreversibly precipitation
Less suitable to antibody-based techniques
What is immersion fixation?
Fresh tissue is immersed in fixing fluid and gently agitated
Preferred for small dissected samples
Large samples less agitation
What is perfusion fixation?
Injection of fixing fluid in animal
The preferred method when possible
Factors that affect the quality of fixation
pH changes
Length of incubation (1mm/hr preferred)
Sample size (<5mm3 preferred)
TEmperature (4ºC less penetration) (room temperature less degeneration)
What is cryopreservation?
Preservation of tissue structure and components by freezing them rapidly without fixation
Name methods of cryopreservation
Snap-freezing the sample with dry ice or liquid nitrogen
Advantages of cryopreservation
Fastest method Minimal changes to protein structure Rapid cooling (-70ºC liquid nitrogen) minimizes the damage of tissue (ice-crystal artifact)
Disadvantages of cryopreservation
Poor morphology
Degradation continues
Requires special cold storage equipment
Do not permanently fixate tissue
