Fixation part 2

Types of Fixative

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1. Aldehydes
2. Oxidizing agents
3. Alcohol based
4. Metallic group of fixatives

Act by cross-linking proteins.

Aldehydes & oxidizing agents

Protein-denaturing agents.

Alcohol-based fixatives

Act by forming insoluble metallic precipitates

Metallic fixatives

Fixatives according to Composition

Simple fixatives and Compound fixatives

Fixatives according to Action

Microanatomical fixatives and Cytological fixatives

Simple fixatives

1. Aldehydes


2. Metallic fixative


3. Picric acid
4. Acetic acid
5. Acetone
6. Alcohol
7. Osmium tetroxide

Aldehydes examples in simple fixative

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1. Formaldehydes
2. Glutaraldehyde

Metallic fixative examples in simple fixative

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1. Mercuric chloride
2. Chromate fixatives

Permit general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of the tissues in question

Microanatomical fixatives

Those that preserve specific parts and particular elements of the cell itself

Cytological fixatives

Microanatomical fixatives examples

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1. 10% formol-saline
2. 10% neutral buffered formalin
3. Heidenhain’s Susa
4. Formol sublimate (Formol corrosive)
5. Zenker’s solution
6. Zenker-formol (Kelly’s solution)
7. Bouin’s solution
8. Brasil’s solution

Preserve nuclear structures

Nuclear fixatives

Cytological fixatives types

1. Nuclear fixatives 
2. Cytoplasmic fixatives
3. Cytological fixatives

Nuclear fixatives contains ________________ as a primary component (pH < 4.6)

Glacial acetic acid

Nuclear fixatives examples

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1. Flemming’s fluid
2. Carnoy’s fluid
3. Bouin’s fluid
4. Newcomer’s fluid
5. Heidenchain’s Susa

Preserves cytoplasmic structures

Cytoplasmic fixatives

Cytoplasmic fixatives should not contain glacial acetic acid which destroys __________________________ (pH > 4.6)

Mitochondria and golgi bodies

Cytoplasmic fixatives examples

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1. Flemming’s fluid without acetic acid
2. Kelly’s fluid
3. Formalin with post-chroming
4. Regaud’s fluid (Muller’s fluid)
5. Orth’s fluid

Those that preserves chemical constituents of cells and tissues

Histochemical fixatives

Cytological fixatives examples

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1. 10% formol-saline
2. Absolute ethyl alcohol
3. Acetone
4. Newcomer’s fluid

It is the process of placing an already fixed tissue in a second fixative

Secondary fixation

Secondary fixation make special staining techniques possible by acting as a

Mordant

Secondary fixation usually done _____________

before dehydration

Secondary fixation is usually done on ______________________

Deparaffinized sections

Secondary fixation is usually done on deparaffinized sections ____________

before staining

A form of secondary fixation where a primarily fixed tissue is placed in an aqueous solution of 2.5 to 3% potassium dichromate to act as a mordant

Post Chromatization

It is the process of removing excess fixative from the tissue after fixation in order to improve staining and remove artifacts from tissues

Washing out

Examples use for washing out

Tap water, 20-57% alcohol and alcoholic iodine

Difficulties Caused by Improper Fixation:

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1. Failure to arrest early autolysis of cells


2. Removal of substances soluble in fixing agent


3. Presence of artifact pigments on tissue sections


4. Tissues are soft and feather-like in consistency


5. Loss or inactivation of enzymes needed for study


6. Shrinkage and swelling of cells and tissue structure


7. Tissue blocks are brittle and hard

Cause of failure to arrest early autolysis of cells

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* Failure to fix immediately
* Insufficient fixative

Cause of presence of artifact pigments on tissue sections

Incomplete washing of fixative

Cause of tissues are soft and feather-like in consistency

Incomplete fixation

What could be the result of wrong choice of fixative

* Removal of substances soluble in fixing agent
* Loss or inactivation of enzymes needed for study

Cause of over fixation

Shrinkage and swelling of cells and tissue structure

Cause of Prolonged fixation

Tissue blocks are brittle and hard

Acetone Fixation

Fix cells in -20°C acetone for 5-10 minutes.

No permeabilization step needed following ___________________

Acetone Fixation

Methanol Fixation

Fix cells in -20°C methanol for 5-10 minutes.

Permeabilization step is needed following _____________________

Methanol Fixation

Ethanol Fixation

Fix cells in cooled 95% ethanol, 5% glacial acetic acid for 5-10 minutes.

Methanol-Acetone Fixation

Fix in cooled methanol, 10 minutes at –20 °C

Remove excess methanol.

Methanol-Acetone Fixation

In Methanol-Acetone Fixation, permeabilize with cooled ______ for 1 minute at –20 °C.

Acetone

Methanol-Acetone Mix Fixation

* 1:1 methanol and acetone mixture.
* Make the mixture fresh and fix cells at -20 C for 5-10 minutes.

Formalin Fixation

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* Fix cells in 10% neutral buffered formalin for 5-10 minutes.
* Rinse briefly with PBS.
* Permeabilize with 0.5% Triton X-100 for 10 minutes.

Paraformaldehyde-Triton Fixation

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* Fix in 3-4% paraformaldehyde for 10-20 minutes.
* Rinse briefly with PBS.
* Permeabilize with 0.5% Triton X-100 for 10 minutes.

Paraformaldehyde-Methanol Fixation

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* Fix in 4% paraformaldehyde for 10-20 minutes.
* Rinse briefly with PBS.
* Permeabilize with cooled methanol for 5-10 minutes at –20 °C.

Fixation Artifacts

1. Formalin pigment
2. "Crush artifact"

A well-known artifact that may be produced under acid conditions.

Formalin pigment

Formalin pigment may be eliminated or reduced by

Fixation in phenol-formalin

May be found in surgical specimens particularly in liver biopsies, associated with an intense eosinophilic staining at the center of the tissue in H&E stained sections.

“Crush artifact”

Two major groups of chemical fixatives:

1. Crosslinking fixatives (aldehydes)
2. Precipitating (or denaturing) fixatives (alcoholic fixatives)

Fixatives that act by creating covalent chemical bonds between proteins in tissue

Crosslinking fixatives (aldehydes)

Fixatives that act by reducing the solubility of protein molecules by disrupting hydrophobic interactions stabilizing protein molecules

Precipitating (or denaturing) fixatives (alcoholic fixatives)

Aldehyde Fixatives

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1. Formaldehyde
2. 10% Formol Saline
3. 10% NBF (Neutral Buffered Formalin)
4. Formol-Corrosive (Formol-Sublimate)
5. Glutaraldehyde
6. Formol-calcium
7. Karnovsky-paraformaldehyde
8. Acrolein

Not routinely used for tissues due to brittleness and hardness.

Precipitating (Alcoholic) Fixatives

Preferred for cytologic smears, quick action, good nuclear detail.

Precipitating (Alcoholic) Fixatives

 Rarely used alone for fixing blocks

Methanol, ethanol, acetone

Precipitating (Alcoholic) Fixatives suitable for studying

Nucleic acids

Physicians use spray can alcohol fixatives for

PAP smears

Preferred for fixing thin layer preparations (blood films, cell cultures).

Methanol

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Alcohol Fixatives

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1. 100% Methyl Alcohol (Methanol)
2. 95% Isopropyl Alcohol
3. Ethyl Alcohol
4. Carnoy’s Fixative
5. Clarke’s Solution
6. Alcoholic Formalin
7. Formol-acetic Alcohol
8. Gendre’s Fixative
9. Newcomer’s Fluid

Employs the use of the metal, mercury

Metallic Fixatives

Increases staining brightness and gives excellent nuclear detail

Metallic Fixatives

Best fixative for hematopoietic and reticuloendothelial tissues

Metallic Fixatives

Metallic fixatives

1. Mercuric chloride


1. Zenker’s solution
2. Zenker-Formol (Helly’s Solution)
3. Lillie’s Solution (B-5 Fixative)
4. Heidenhain’s Susa Solution
5. Schauddin’s Sublimated Alcohol
2. Chromate fixatives


1. Chromic acid
2. Potassium Dichromate
3. Regaud’s (Muller’s Fluid)
4. Orth’s Fluid
3. Lead fixatives

Includes permanganate fixatives, potassium dichromate, chromic acid and osmium tetroxide

Oxidizing Agents

Oxidizing Agents used mainly as a

Secondary fixative

Oxidizing Agents

1. Osmium Tetroxide (OsO4) or Osmic acid
2. Flemming’s Solution

An explosive hazard in dry form

Picric Acid Fixatives

It is a yellow colored solution which also dyes the tissues.

Picric Acid Fixatives

It acts as a fixative and a dye at the same time

Picric Acid Fixatives

Picric Acid Fixatives preserved

Glycogen

T/F: Picric acid fixed tissues must never be washed in water before dehydration

True

Picric Acid Fixatives should be transferred or washed directly with

70% alcohol 

Picric Acid Fixatives

1. Bouin’s Solution
2. Hollande’s Solution
3. Gendre’s Solution

It is better and less “messy” than Bouin’s solution

Brasil’s Alcoholic Picroformol Fixative

It is an excellent fixative for glycogen

Brasil’s Alcoholic Picroformol Fixative

They are used in 4% aqueous solution of basic lead acetate

Lead Fixatives

It is recommended for acid mucopolysaccharides

Lead Fixatives

It is used for the precipitation of proteins and nucleic acids

Trichloroacetic acid

It is used as a fixative and a weak decalcifying agent

Trichloroacetic acid

Not recommended as morphological fixative for tissue blocks

Acetone

It is primarily used in fixing brain tissues for the diagnosis of rabies

Acetone

It is a simple salt solution which provides a stable transport medium of a fresh unfixed tissues such as renal, skin and oral mucosa biopsies

Michel’s Solution

T/F: Michel’s Solution is a fixative

False

Slow freezing of unfixed tissues near 0℃ must be avoided since this may promote formation of

Ice crystal artifacts

Small tissue biopsies may be placed in a petri dish with moistened filter paper to prevent

Drying

If placed in normal saline solution (NSS) during the operation, what could occur before fixation is carried on?

Autolysis

Tissues should not be more than 5 mm. thick except in

Lung edema

Lung edema tissue slice may be __________ thick

1-2 cm

The amount of fixative must be adequate, approximately 10-20 times the volume of the tissue specimen except in

Osmium tetroxide

Osmium tetroxide requiring only _____________ that of tissue volume for fixation.

5-10 times

For prolonged fixation (e.g. museum preparation) volume of fixing fluid should not be _____________ that of the tissue.

less than 50 times

In most instances, fixed tissues must be washed thoroughly to remove _______________ before staining.

Salts and/or pigments

Hollow organs (e.g. stomach, intestines) should be __________________________ before being immersed in adequate fixing solution.

packed with cotton soaked in fixative or completely opened

Human brains may be suspended by a cord tied under the ________________ to prevent flattening.

Circle of Willis

Dense tissues such as brains are poorly penetrated, hence require long fixation usually ______________ prior to sectioning

2 weeks

Frozen sections may lead to formation of ________________ artifacts

Ice crystal