2010 Lab 3

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Contents

Preparation/Fixation

This lab is introduction to histological techniques and tissue/cell fixation. The lab will also introduce 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. Later analysis and immunhistochemistry will be covered in a future Laboratories.

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.

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)

Objectives

  • Brief understanding of chemical OHS issues
  • Brief understanding of histological staining techniques
  • Brief understanding of tissue preparation and sectioning
  • Understanding of fixation techniques

Occupational Health and Safety (OHS)

  • School of Medical Sciences, Occupational Health and Safety (OHS) Committee
    • "To facilitate a safe work environment by developing and documenting OHS programs to coordinate training of staff and students and by overseeing the implementation of OHS procedures and policies in the School of Medical Sciences."
  • Australian Acts and Standards
  • An OHS 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


Material Safety Data Sheets (MSDS)

  • Material Safety Data Sheets (MSDS) are 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 MSDSs.
    • 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 MSDS's for their own reference.
  • There is currently no coordinated international standard and different countries may have different requirements.


MSDS 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: 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

  • cells are preserved and hardened by rapid freezing
  • Used in surgical biopsies of tissue
  • advantages and disadvantages
    • 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

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.

Tissue Embedding

Cell cultures

  • Cell cultures are usually only a layer or two of cells thick and are generally not embedded in a support media, except for electron microscopic (EM) preparation.
  • This tissue thickness also means that fixation can be quite rapid.

Paraffin Embedding

Automatic wax embedding machine
  • Paraffin waxes can allow easy long-term tissue storage and ease of sectioning by supporting the tissue during cutting.
  • Often requires a large number of steps in fixation, series of steps for embedding, sectioning and finally removal of embedding matrix for staining.
  • There are automated paraffin embedding systems that remove many of the preparation steps.
  • Can sometimes not be suitable for immunochemistry fluorescence techniques.

Cryoembedding

  • Possible freezing artifact, ice crystal formation if not controlled chilling. Freezing can be critical.
    • vapor phase of liquid nitrogen
    • thawing isopentane
  • OCT (Optimal Cutting Temperature) commercial Cryo Embedding Medium
  • Not suitable for large amounts, by volume) of tissue (usually 0.5 cm x 0.5 cm x 0.5 cm max)

Links: Face down cryoembedding technique

References

Textbooks

Essential Cell Biology

  • Chapter

Molecular Biology of the Cell

Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter New York and London: Garland Science; c2002


Molecular Cell Biology

Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. New York: W. H. Freeman & Co.; c1999

The Cell- A Molecular Approach

Cooper, Geoffrey M. Sunderland (MA): Sinauer Associates, Inc.; c2000

Search Online Textbooks

Books

PubMed

  • PubMed is a service of the U.S. National Library of Medicine that includes over 18 million citations from MEDLINE and other life science journals for biomedical articles back to 1948. PubMed includes links to full text articles and other related resources. PubMed
  • PubMed Central (PMC) is a free digital archive of biomedical and life sciences journal literature at the U.S. National Institutes of Health (NIH) in the National Library of Medicine (NLM) allowing all users free access to the material in PubMed Central. PMC
  • Online Mendelian Inheritance in Man (OMIM) is a comprehensive compendium of human genes and genetic phenotypes. The full-text, referenced overviews in OMIM contain information on all known mendelian disorders and over 12,000 genes. OMIM
  • Entrez is the integrated, text-based search and retrieval system used at NCBI for the major databases, including PubMed, Nucleotide and Protein Sequences, Protein Structures, Complete Genomes, Taxonomy, and others Entrez

Search Pubmed

Reviews

  • Sample preparation for scanning electron microscopy of plant surfaces--horses for courses. Pathan AK, Bond J, Gaskin RE. Micron. 2008 Dec;39(8):1049-61. Epub 2008 May 27. Review. PMID: 18586502
  • Correlated light and electron microscopy of the cytoskeleton. Auinger S, Small JV. Methods Cell Biol. 2008;88:257-72. Review. PMID: 18617038
  • Tissue microdissection. Erickson HS, Gillespie JW, Emmert-Buck MR. Methods Mol Biol. 2008;424:433-48. Review. PMID: 18369881

External Links

Cell Biology Applications of Fluorescence Microscopy Stephen Rogers Histotechniques

2010 Course Content

Lectures: Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cell Mitochondria | Cell Junctions | Cytoskeleton Introduction | Cytoskeleton 1 Intermediate Filaments | Cytoskeleton 2 Microtubules | Cytoskeleton 3 Microfilaments | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells 1 | Stem Cells 2 | Development | Revision

Laboratories: Introduction to Lab | Microscopy Methods | Preparation/Fixation | Immunochemistry | Cell Knockout Methods | Cytoskeleton Exercise | Confocal Microscopy | Microarray Visit | Tissue Culture 1 | Tissue Culture 2 | Stem Cells Lab | Stem Cells Analysis

Dr Mark Hill 2013, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G