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 alnalysis and immunhistochemistry will be covered in a future Lab.
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)
Page Links: Introduction | Objectives | Paraformaldehyde | Formalin | Methanol | Detergents | MSDS
Brief understanding of histological staining techniques
Brief understanding of tissue preparation and sectioning
Understanding of fixation techniques
Brief understanding of chemical OHS issues
Aldehydes fix tissue by introducing cross-links between different tissue components (proteins, nucleic acids, lipids). The degree of cross-linking produced in a tissue is also proportional to fixation time. Cross-links are generated between several reactive groups (mainly -NH2 groups) such as found in protein lysine residues.
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
Google search: paraformaldehyde fixation MSDS: Paraformaldehyde MSDS | Paraformaldehyde MSDS (PDF)
Methanol dehydrates, coagulates and precipitates cellular proteins, nucleic acids and carbohydrates.
The process involves no covalent bonding between methanol fixative and tissue components.
Can be combined with an acetone step.
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.
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
Note that while information found on internet chemical MSDS pages may be very similar, international sites may not conform to Australian Worksafe format.
National Occupational Health and Safety Commission | Workcover NSW
Detergents are not really "fixative", but a number of different types are often used in the fixation process as a method of preserving or accessing antigenic sites that may be blocked or effected by the fixation process itself.
The 2 major classes are the ionic detergents and nonionic detergents.
Beverly Faulkner-Jones- Fixation, processing, sectioning and storage of tissues
Search all NCBI Books for the term "fixation"
Search MBoC4 for the term "fixation"
Tissues Are Usually Fixed and Sectioned for Microscopy (&rid=mboc4.section.1715#1733)These commercial links are for educational purposes only and do not reflect endorsement of a specific product.
MSDS Online search Approx 10,000 sheets are added each month. Free registration/login is required to view MSDSs. OSHA, DOT, ANSI and WHIMIS compliant sheets in PDF format.
Vermont SIRI Either select a letter A-Z to browse manufacturers alphabetically (for sheets not in the SIRI collection) or do a full-text keyword search in the SIRI MSDS database. Mostly OSHA-style sheets,all in text file format. Lots of additional safety links and info.
Online Mendelian Inheritance in Man (OMIM) provides background information to cell proteins and their relationship to known diseases. The introduction of each entry gives a historic background to the disease/protein and links to key references.
Genes & Disease Prepared by NCBI to give a "snapshot" overview of a few of the most common genetic diseases.
National Library of Medicine (USA) Bookshelf has a number of excellent online textbooks that can be all be searched for general background information using the window at the top of the linked page.
UNSW Cell Biology textbooks links to NLM cell biology textbooks and the publishers sites which often contain additional online resources.
Different databases can be selected from the pulldown menu.
Selected option "Entrez" shows the results from searching all the different NCBI databases simultaneously.
NCBI "Established in 1988 as a national resource for molecular biology information, NCBI creates public databases, conducts research in computational biology, develops software tools for analyzing genome data, and disseminates biomedical information - all for the better understanding of molecular processes affecting human health and disease"
This allows a Search of NIH Medlineplus Medical Dictionary. Type the word that you would like to find. If unsure of spelling, type the first few letters, followed by an asterisk(*).
microscopy, tissue culture, cell fractionation, histology, immunochemistry, protein separation,
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This Lab is an introduction to your Group Project.
The UNSW Library online or the National Library of Medicine search will provide the most scientific resources. ABC searches will provide some "news" relevance and Google image searches will show pictorial images that relate to your topic.
Please email Dr Mark Hill if you wish to make a comment about this current project.