CHAPTER 7: CHEMICAL FIXATIVES PART 1 Flashcards
- Aldehydes
- Act by creating covalent chemical bonds between proteins in tissue
- Anchors soluble proteins to the cytoskeleton, and lends additional rigidity to the tissue
Crosslinking Fixatives
- Commonly used cross-linking fixative
- Good for immunohistochemical techniques
- Long term storage and good tissue penetration
- Vapor form is used for cell smears
Formaldehyde
Standard solution of Formaldehyde
10% neutral buffered formalin / 3.7%-4.0% formaldehyde in phosphate buffered saline
Made with formaldehyde but the percentage denotes a different formaldehyde concentration
Formalin
10% neutral buffered formalin is equivalent to __% formaldehyde
4
- Most widely used fixative for routine histology
- Buffered to pH 7 with phosphate buffer
- Immunohistochemistry and interphase FISH
- Contains 10% methanol to retard the formation of higher polymers that eventually fall out of solution as paraformaldehyde (deposits of white powder)
10% Neutral buffered formalin
Routine fixation with 10% Neutral buffered formalin
- Dissect specimen ASAP and immerse in a large volume of fixative
- Place at 4 degC and fix overnight
- Wash tissue well in several changes of Phosphate Buffered Saline (PBS); tissue may be stored in cold PBS for short period of time (2 or 3 days) and will be safe in ethanol since there is no danger of bacterial degradation
- Should not be stored in 70% ethanol for extended period; instead, store in PBS with sodium azide
Fixation time of small tissues (10x10x3) in 10% Neutral buffered formalin
a. 12-24 hours
b. 8-12 hours (overnight)
c. 24 hours
d. 2-4 weeks
a. 12-24 hours
T/F: Fixation with formaldehyde is largely complete within 24 hours, although cross-linking still occurs for at least 2 weeks
True
T/F: At temperatures normally used for fixation (20-22°C), native DNA and RNA react with formaldehyde.
False (do not react)
If reaction mixtures are heated at about 45 deg C (RNA) and 65 deg C (DNA), reaction begins due to _______.
uncoiling
T/F: Only at elevated temperatures used when tissues are infiltrated with paraffin or resin, can a reaction with any remaining fixative take place.
True
Advantages of 10% Neutral buffered formalin
- It is cheap, readily available, easy to prepare, and relatively stable, especially if stored in buffered solution.
- It is compatible with many stains, and therefore can be used with various staining techniques depending upon the need of the tissues
- It does not over-harden tissues, even with prolonged periods of fixation, as long as solutions are regularly changed.
- It penetrates tissue well
- It preserves fat and mucin, making them resistant to subsequent treatment with fat solvents, and allowing them to be stained for demonstration
- It preserves glycogen
- It preserves but does not precipitate proteins, thereby allowing tissue enzymes to be studied. It does not make tissues brittle and is therefore recommended for nervous tissue preservation.
- It allows natural tissue colors to be restored after fixation by immersing formalin-fixed tissues in 70% alcohol for one hour and is therefore recommended for colored tissue photography.
- It allows frozen tissue sections to be prepared easily.
- It does not require washing out, unless tissues have stayed in formalin for excessively long periods of time
If unbuffered, Formalin reduces neutrophilic and eosinophilic staining of cells. It also forms abundant brown pigment granules on blood-containing tissues, e.g., spleen, due to blackening of hemoglobin.
False/True
True/False
Both True
Both False
False/True (basophilic and eosinophilic)
Prolonged fixation may produce:
a. Bleaching and loss of natural tissue colors
b. Fat dispersal into fluid
c. Loss of glycogen and urate crystals
AOTA
FACTORS THAT INFLUENCE FORMALIN FIXATION
1) Post-Mortem / Post-Surgical Interval
2) Composition of Fixative
3) Volume of Fixative
4) Fixation Time (24 hours in NBF)
5) Temperature
6) Tissue Thickness (3-5mm)
7) Post-Fixation Storage (for necessary delay, 3 days in the cold in 70% ethanol)
Mixture of fixatives that is useful for electron cytochemistry
Karnovsky’s paraformaldehyde- glutaraldehyde
-Mixture of fixative
- Rapid, preserve morphology and enzyme activity at low concentration
- Used for immersion fixation of surgical biopsies
Acrolein
remove white paraformaldehyde deposits
10% methanol
If added to formaldehyde, it prevents decomposition to formic acid or precipitation to paraformaldehyde (but unsuitable for EM due to protein denaturation)
Methanol
Change fluid fixative every _____ to prevent bleaching of tissues
3 months
Immerse tissues in _______ after fixation to restore its natural colors
70% alcohol
T/F: Saturated alcoholic picric acid or 1% potassium hydroxide in 80% alcohol can remove brown/black crystalline precipitate formed by formic acid with blood.
True
______ can be added to prevent dispersion of fat into fluid
Cadmium/Cobalt
T/F: Magnesium carbonate or calcium can buffer/neutralize acid reaction due to formic acid formation to prevent explosion
True
T/F: Cadmium acetate can buffer formalin without leaving deposits on the tissue parts exposed to it
False (leave deposits)
Secondary fixation of formalin-fixed tissues (1-2 hours) in Helly’s fluid for _______ or in _______ for 4-16 hours to improve staining and produce firmer and harder consistency.
- 4-6 hours
- Formol sublimate
T/F: Prevent deposition of hematin from acidic formalin by maintaining alkaline pH
False (neutral)
T/F: Wash tissue in demineralized water if post-fixed in osmic acid to prevent hypotonicity and bleaching
False (Do not wash in demineralized water)
Fixation of tissue blocks less than or equal to ______ thickness is usually complete in 6-12 hours at room temperature
5 mm
pH of 10% Formal-Saline
6.8
Widely used prior to introduction of Phosphate Buffered Formalin
10% Formal-Saline
Fixation time of 10% Formal-Saline
12-24 hours
- Central nervous tissues and general post-mortem tissues for histochemical examination
- Preservation of lipids (phospholipids)
10% Formal-Saline
Advantages of 10% Formal-Saline
- It penetrates and fixes tissues evenly.
- It preserves microanatomic and cytologic details with minimum shrinkage and distortion.
- Large specimens may be fixed for a long time provided that the solution is changed every three months.
- It preserves enzymes and nucleoproteins.
- It demonstrates fats and mucin.
- It does not over-harden tissues, thereby facilitating dissection of the specimen.
- It is ideal for most staining techniques, including silver impregnation
- It allows natural tissue color to be restored upon immersion in 70% alcohol.
Advantages of 10% Formal-Saline
- It penetrates and fixes tissues evenly.
- It preserves microanatomic and cytologic details with minimum shrinkage and distortion.
- Large specimens may be fixed for a long time provided that the solution is changed every three months.
- It preserves enzymes and nucleoproteins.
- It demonstrates fats and mucin.
- It does not over-harden tissues, thereby facilitating dissection of the specimen.
- It is ideal for most staining techniques, including silver impregnation
- It allows natural tissue color to be restored upon immersion in 70% alcohol.
T/F: 10% Formal-Saline is a rapid fixative
False (slow; 24 hours or longer)
Formal-saline fixed tissues tend to shrink during alcohol dehydration; this may be reduced by __________
secondary fixation
T/F: Acid dye stains more brightly than when fixed with mercuric chloride
False (less brightly)
One of the advantages of formol saline is that the 4. metachromatic reaction of amyloid is reduced.
False (disadvantage)
pH of 10% Neutral-Buffered Formalin
7.4
Best fixative for tissues containing iron pigments and for elastic fibers which do not stain well after Susa, Zenker’s or chromate fixation
10% Neutral-Buffered Formalin
T/F: 10% Neutral-Buffered Formalin requires no post-treatment after fixation and goes directly into 80% alcohol for processing
True
T/F: 10% Neutral-Buffered Formalin requires no post-treatment after fixation and goes directly into 80% alcohol for processing
True
Disadvantages of 10% Neutral-Buffered Formalin
- It is longer to prepare; hence, is time-consuming.
- Positivity of mucin to PAS is reduced.
- It may produce gradual loss in basophilic staining of cells
- Reactivity of myelin to Weigert’s iron hematoxylin stain is reduced.
- It is inert towards lipids, especially neutral fats and phospholipids.
The following statements about Unbuffered Zinc Formalin are true except for:
- Devised as alternatives to mercuric chloride formulations
- Said to give improved results with immunohistochemistry
- Zinc sulphate is more corrosive than zinc chloride
a. 1
b. 2
c. 3
c. 3
Fixation time of Formol-Corrosive (Formol-Sublimate)
3-24 hours
Fixation time of Formol-Corrosive (Formol-Sublimate)
3-24 hours
Recommended for routine post-mortem tissues
Formol-Corrosive (Formol-Sublimate)
It is excellent for many staining procedures including silver reticulum methods
Formol-Corrosive (Formol-Sublimate)
It is excellent for many staining procedures including silver reticulum methods
Formol-Corrosive (Formol-Sublimate)
Cytological structures and blood cells are well preserved. There is no need for “washing-out”. Tissues can be transferred directly from fixative to
alcohol.
a.10% Formal-Saline
b.10% Neutral-Buffered Formalin
c. Formol-Corrosive (Formol-Sublimate)
c. Formol-Corrosive (Formol-Sublimate)
It fixes lipids, especially neutral fats and
phospholipids
Formol-Corrosive (Formol-Sublimate)
Disadvantages of Formol-Corrosive (Formol-Sublimate)
- Penetration is slow; hence, tissue sections should not be more than 1 cm thick.
- It forms mercuric chloride deposits.
- It does not allow frozen tissue sections to be made.
- It inhibits the determination of the extent of tissue decalcification.
- Suited for paraffin embedding and sectioning, and immunocytochemical analysis
- Allows for subsequent immune-detection of certain antigens and should be therefore used when objective is to study morphology and protein expression simultaneously
Paraformaldehyde
Polymerized form of formaldehyde, usually obtained as fine white powder; depolymerizes to formalin when heated
Polymerized form of formaldehyde, usually obtained as fine white powder; depolymerizes to formalin when heated
Paraformaldehyde
4% Paraformaldehyde-1% Glutaraldehyde in 0.1M Phosphate Buffer
Karnovsky’s Fixative
- Suitable for use when preparing samples for LM in resin embedding and sectioning, and for EM
- Should always be prepared fresh
Karnovsky’s Fixative
- Made up of 2 formaldehyde residues, linked by a 3-carbon chain
- Causes rapid and irreversible changes, fixes quickly, well suited for EM
- It fixes well at 4 deg C and it gives best overall cytoplasmic and nuclear detail
Glutaraldehyde
Causes deformation of alpha-helix structure in
proteins (not good for immunohistochemical staining)
Glutaraldehyde
most efficient aldehyde blockers
Ethanolamine and lysine
4% Glutaraldehyde is used for small tissue fragments and needle biopsies (2-4 hours @ RT). 2.5% Glutaraldehyde is used for larger tissues less than 4mm thick (6-8 hours up to 24 hours)
a. FALSE/TRUE
b. TRUE/FALSE
c. BOTH TRUE
d. BOTH FALSE
d. BOTH FALSE
Advantages of Glutaraldehyde over Formalin
- It has a more stable effect on tissues, giving a firmer texture with better tissue sections, especially of central nervous tissues.
- It preserves plasma proteins better.
- It produces less tissue shrinkage.
- It preserves cellular structures better; hence, is recommended for electron microscopy.
- It is more pleasant and less irritating to the nose.
- It does not cause dermatitis.
Advantages of Glutaraldehyde over Formalin
- It has a more stable effect on tissues, giving a firmer texture with better tissue sections, especially of central nervous tissues.
- It preserves plasma proteins better.
- It produces less tissue shrinkage.
- It preserves cellular structures better; hence, is recommended for electron microscopy.
- It is more pleasant and less irritating to the nose.
- It does not cause dermatitis.
Advantages of Glutaraldehyde over Formalin
- It has a more stable effect on tissues, giving a firmer texture with better tissue sections, especially of central nervous tissues.
- It preserves plasma proteins better.
- It produces less tissue shrinkage.
- It preserves cellular structures better; hence, is recommended for electron microscopy.
- It is more pleasant and less irritating to the nose.
- It does not cause dermatitis.
Disadvantages of Glutaraldehyde over Formaldehyde
- It is less stable.
- It penetrates tissues more slowly.
- It tends to make tissue (i.e., renal biopsy) more brittle
- It is more expensive.
- It reduces PAS positivity of reactive mucin. This may be prevented by immersing glutaraldehyde- fixed tissues in a mixture of concentrated glacial acetic acid and aniline oil.
