Histology Fixatives

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Introduction

It is critical to match the method of fixation with the intended analytical technique. Some types of analysis are totally incompatible with certain fixation techniques and always consider that "artefacts" can be introduced by the fixation process. Fixation methods discussed on this page include: fresh frozen, precipitation and aldehyde cross-linking. Always consider the Occupational Health and Safety (OHS) issues in relation to chemicals used in this process. More information is available from the School of Medical Sciences OHS webpage.


This content is covered in a practical lab as part of an undergraduate science cell biology course.

In general the Fixation process should:

  1. Preserve cell structure by prevention of tissue autodigestion (autolysis)
  2. Inhibits bacterial and fungal growth (preserves)
  3. Make the tissue resistant to damage during subsequent processing (hardy)


Histology Links: Stains | Fixatives | Menstrual Cycle | Placenta | Heart | Liver | Pancreas | Gall Bladder | Colon | Renal | Respiratory Histology | Bone | Category:Histology | UNSW Histology
Histology Glossary: A | B | C | D | E | F | G | H | I | J | K | L | M | N | O | P | Q | R | S | T | U | V | W | X | Y | Z | ANAT2241 Support | Histology | Histology Stains | Embryology Glossary


Health and Safety (HS)

  • School of Medical Sciences, Health and Safety (H&S) Committee
    • "To facilitate a safe work environment by developing and documenting H&S programs to coordinate training of staff and students and by overseeing the implementation of H&S procedures and policies in the School of Medical Sciences."
  • Australian Acts and Standards
  • An H&S Management System (OHSMS) is a set of plans, actions and procedures to systematically manage health and safety in the workplace that is actively endorsed by a committed employer to achieve:
    • Provision of a safe and health workplace and the prevention/reduction of illness and injury equally for employees and contractors.
    • Identification of workplace hazards, assessment and control of all risks.
    • Active involvement in health and safety matters by managers, supervisors and employees and their representatives.
    • Provision of information and training for employees at all levels so they can work safely.
    • Audit and review of the OHSMS.
    • UNSW OHS Management System

Safety Data Sheets (SDS)

  • Safety Data Sheets (SDS) replace the original term and classification Material Safety Data Sheets (MSDS)
    • Updated as part of "Globally Harmonized System of Classification and Labelling of Chemicals (GHS)"
  • A set of standardised safety information prepared for each of the chemicals used within the laboratory.
  • Each research laboratory is required to keep either a hardcopy or electronic copy of these MSDS's available within the laboratory.
  • Before carrying out any new research technique, in particular for students, should be taken through the location and use of SDSs.
    • the risks and hazards involved with specific chemicals.
    • the correct storage, handling, labeling and disposal of each chemical.
    • ideally they should keep an electronic copy or link to each of these SDS's for their own reference.
  • There is currently no coordinated international standard and different countries may have different requirements.

SDS must state:

  1. a hazardous substance's product name
  2. the chemical and generic name of certain ingredients
  3. the chemical and physical properties of the hazardous substance
  4. health hazard information
  5. precautions for safe use and handling
  6. the manufacturer's or importer's name, Australian address and telephone number.

Note that while information found on internet chemical MSDS pages may be very similar, international sites may not conform to Australian Worksafe format.

Links: Safe Work Australia - GHS | United Nations - GHS | UNSW Chem Alert | USA - NIOSH Pocket Guide to Chemical Hazards

Universal Precautions

  • When dealing with biological materials, in particular human specimens, are a set of precautions designed to prevent transmission of human immunodeficiency virus (HIV), hepatitis B virus (HBV), and other bloodborne pathogens when providing first aid or health care. These precautions should also be used when carrying out basic research on these tissues.
  • Universal precautions involve the use of protective barriers (PPE, personal protective equipment) such as gloves, gowns, aprons, masks, or protective eyewear, which can reduce the risk of exposure of the health care worker's skin or mucous membranes to potentially infective materials. In addition, under universal precautions, it is recommended that all health care workers take precautions to prevent injuries caused by needles, scalpels, and other sharp instruments or devices.


Links: CDC Universal Precautions for Prevention of Transmission of HIV and Other Bloodborne Infections

Three Main Techniques

  1. Fresh Frozen
  2. Precipitation
  3. Aldehyde Cross-linked

Fresh Frozen

File:Cryostat movie label.jpg
Cryostat sectioning
  • Used in surgical biopsies of tissue and research - cells are preserved and hardened by rapid freezing
  • Advantages - rapid processing, retention of some enzyme and protein function, retention of epitopes, retention of fat
  • Disadvantages - requires a cryotome (freezing microtome) for sectioning, thicker sections (8+ micrometers), tissue distortion with cutting, thawing can degrades tissue
Cryostat Cryostat sectioning
Cryostat machine Cryostat Movie

Small Movie | Medium Movie


Links: frozen section technique | Video - frozen sectioning

Precipitation

  • Immersion in cooled organic solvents- methanol or acetone or acids
  • Acidic precipitation does not preserve cellular structures well, rarely used (except for specific protocols, such as mitotic chromosome spreads)
  • Fixation by precipitation does not preserve the three-dimensional organization of specimens, therefore not recommended for confocal microscopy.
  • Cultured cells fixed with cold methanol shrink by as much as 50%.
  • Advantages- speed -(fixation usually taking a few minutes), retention of epitopes (antibody binding sites) not covalently modified as they might be with aldehyde fixation,

simultaneous permeabilization of cellular membranes (no need for detergent-treatment), precipitation will not introduce autofluorescence

(Text modified from Cell Biology Applications of Fluorescence Microscopy by Stephen Rogers)

Methanol

  • precipitation fixation
  • Methanol dehydrates, coagulates and precipitates cellular proteins, nucleic acids and carbohydrates
  • The process involves no covalent bonding between methanol fixative and tissue components

Chloroform-containing Fixative

  • Carnoy’s fixative
  • rapid tissue penetration (small tissue pieces in minutes not hours)
  • can damage tissues when transferred from aqueous solution (extreme hydrophobicity of chloroform and rapid dehydration)

Fixative components

  • Chloroform 30%
  • Ethanol (100%) 60%
  • Acetic Acid (Glacial) 10%

Aldehyde Cross-Linked

  • Aldehydes form covalent bonds between adjacent amine-containing groups through a Schiff acid-base reaction.
  • Cross-links are generated between several reactive groups (mainly -NH2 groups) such as found in protein lysine residues.
  • good fixatives for proteins and nucleic acids.
  • most commonly used aldehydes are formaldehyde (formalin), paraformaldehyde and glutaraldehyde
  • The degree of cross-linking produced in a tissue is also proportional to fixation time.
  • Aldehydes are suspected carcinogens, to be used only in well-ventilated areas or fume hoods and contact with skin or eyes avoided

Formalin

  • Aldehyde Cross-Link fixation
  • Formalin is a 37% aqueous solution of formaldehyde, which fixes by cross-linking like other aldehyde fixatives and is suitable for most histological purposes
  • Neutral buffered formalin (fixation time 12-24 hours) is preferred to formol-saline (a single 10% solution of formalin in 9% aqueous NaCl) as formalin pigment is avoided
  • Specimens may be stored in this fluid and the solution is isotonic.
  • Can be combined with a precipitation step (acetone etc) for permeabilization
  • Synonyms: bvf, FA, fannoform, formalith, formalin, formalin 40, formic aldehyde, formol, fyde, hoch, karsan, lysoform, methyl aldehyde, methylene glycol, methylene oxide, methanal, morbicid, oxomethane, oxymethylene, paraform, polyoxymethylene glycols, superlysoform
  • Molecular formula: CH2O CAS No: 50-00-0 MSDS: Formaldehyde MSDS

Paraformaldehyde

  • Aldehyde Cross-Link fixation
  • Used generally fresh
  • generates less fluorescent artifacts than formaldehyde

Uses: immunochemistry, in situ hybridization, cell staining

Synonyms: paraform, polyoxymethane, polymerised formaldehyde, alacide, flo-mor, formagene

Molecular formula: (CH2O)n CAS No: 30525-89-4

Gluteraldehyde

  • Aldehyde Cross-Link fixation


Other Fixation Considerations

Detergents

  • Detergents are not really "fixative", but a number of different types are often used in the fixation process.
  • Detergents can selectively remove components from the material to be fixed or already fixed, as a method of preserving or accessing antigenic sites that may be blocked or effected by the fixation process itself.
  • The 2 major detergent classes
    • ionic detergents
    • nonionic detergents

Osmolality

  • Generally a phosphate buffered saline (PBS) is used but wil differ for some specific fixatives. Changes in osmolality can affect tissue structure and introduce artefacts.
    • hypertonic solutions may cause cells to shrink.
    • hypotonic solutions may cause the cells may swell and burst.


Bouin

Bouin’s Fixative (Bouin’s Fluid, Bouin solution)

Bouin’s fixative is the primary mordant solution, substance used to set a dye, in the trichrome stains. It is a compound fixative composed of picric acid, acetic acid and formaldehyde in an aqueous solution. Picric acid acts to balance the fixation effects of the other 2 agents.

  • Acetic acid tissue swelling effect is balanced by the tissue shrinking effect of picric acid.
  • Formalin causes cytoplasm to become basophilic effect is balanced by the picric acid. Good for nuclear and cytoplasmic (Stain - Haematoxylin Eosin) staining.
  • Formalin causes tissue hardening effect is balanced by the soft tissue fixation of picric acid.
  • Picric acid when retained interferes with some stains, including those used for blood cells.

Whole specimen fixation

  • small specimens for at least 6 hours.
  • other specimens can be fixed up to two days.
  • large specimens can be fixed for up to three days.

Do not use Bouin’s to fix before in situ hybridisation, as then unable to detect RNA.

Formalin

Kaiserling

(Kaiserling’s preservative, Kaiserling’s fixative) An aqueous solution of potassium nitrate, potassium acetate, and formalin. There are four preservative variants (Kaiserling 1896, 1899, Kaiserling I and II), two are used in anatomy museums (Kaiserling I and II). Johann Carl Kaiserling (1869 - 1942) was a German pathologist.




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Cite this page: Hill, M.A. 2017 Embryology Histology Fixatives. Retrieved February 28, 2017, from https://embryology.med.unsw.edu.au/embryology/index.php/Histology_Fixatives

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© Dr Mark Hill 2017, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G