HIS Fixation, Pigments, and Artifacts Flashcards
(48 cards)
Functions of fixatives (3)
- Prevent autolysis
- Stabilize tissue morphology (carbs/ lipids)
- Enhance staining
2 mechanisms of fixation
- Denaturation
- Formation of cross-links
5 ways fixatives can Denature proteins
- Aldehydes
- Alcohols
- Acids
- Heavy metals
- Heat
Why may heat fixation not be preferred over chemical fixation ?
It produces random protein structures = reproducibility issues when analyzing tissue
How do alcohols denature proteins ?
- alcohols are hydrophilic = removes water from protein = exposed hydrophobic domains unfolds the peptide
- disrupts hydrogen bonding = new OH bonds form between alcohol and amino acids = stabilize denatured protein structures and harden tissue
Fixation __ proteins
Fixation DENATURES proteins
How do Cross-linking fixatives fix tissues ?
Stabilizes protein by forming methylene bridges = hardens tissue with minimal shrinkage
Additive fixatives
Chemically BIND with tissue (components) to alter primary, secondary, and tertiary structure:
- aldehydes
- oxides (osmium)
- heavy metals (mercury, zinc)
- acids
Non-additive fixatives
Do not bind to tissue and only alter tertiary structure:
- alcohols (ethanol, methanol, acetone)
List factors that affect fixation (4):
- Temperature
- Tissue thickness/ size
- Time
- Fixative volume
How does temperature affect fixation ?
- warmer = faster fixation
- routine light microscopy <45°C; TYPICALLY RT
- electron microscopy <37°C
How does tissue thickness/ size affect fixation ?
- Fixative will penetrate at different rate
<4mm thick in formalin
<1mm thick in glutaraldehyde
How does time affect tissue fixation ?
- transport should be minimized to prevent putrefaction
- neutral buffered formalin = minimum of 8 hours
- breast = minimum of 24 hours
How does fixative volume affect fixation ?
(At least 20 TIMES ) higher ratios of fixative:specimen volume are better
Neutral buffered formalin contains _% formaldehyde
Neutral buffered formalin contains ~4% formaldehyde
Why may manufacturers add 10% methanol to concentrated formaldehyde ?
To prevent formation of PARAFORMALDEHYDE; a white precipitate
Formalin: what kind of fixative? Shrinkage ? Hardening ?
- additive, non-coagulant
- cross-links with positive amino acids
- less shrinkage than other fixatives
- extensively hardens tissues over time
Describe fixation by formalin
Since formalin is an aldehyde:
1. RAPID penetration of tissue and binding of positive amino acids
2. SLOW formation of Cross-links = methylene bridges of adjacent proteins = stabilized protein structure (takes days)
Why is formalin the fixative of choice despite its toxicity ?
Cheap, stable and available for other special stains
How does glutaraldehyde differ from formaldehyde (formalin) ?
- has two aldehydes (think shark face) instead of one
- cross-linking occurs simultaneously as penetration BUT
- cross-linking slows down penetration
Glutaraldehyde: What kind of fixative ? Shrinkage ? Hardening ?
- additive, non-coagulant fixative used in electron microscopy
- unstable; must be used fresh and buffered to prevent becoming acidic
- minimal shrinkage; hardens tissue
Since cross-linking slows down further penetration in glutaraldehyde, tissue must be __.
Since cross-linking slows down further penetration in glutaraldehyde, tissue must be 1mm thin ! (Faster penetration)
NOTE: used in ELECTRON MICROSCOPY
Acetic acid: What kind of fixative ? Shrinkage ? Hardening ?
- coagulant (precipitates DNA + preserves nucleoproteins)
- CANNOT fix cytoplasmic proteins; MIXED IN A COMPOUND FIXATIVE
- swells tissue the most (can counteract shrinkage)
- does not harden tissue
Picric acid storage/ handling requirements
- must be stored WET; wipe up small spills before they dry = dry picric acid is explosive
- NEVER POUR DOWN DRAIN = can combine with metal pipes = picrate salts are explosive even when wet ! womp womp
