2015 Lab 3

From CellBiology

Preparation/Fixation

Blood Cells (fixed and stained)

This lab is introduction to histological techniques and tissue/cell fixation. The lab will also introduce Health and Safety (H&S) issues in relation to chemicals used in this process. More information is available from the School of Medical Sciences H&S 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)
  4. Allow access of stains and antibodies (permeable)

Objectives

  • Brief understanding of chemical H&S issues
  • Brief understanding of histological staining techniques
  • Brief understanding of tissue preparation and sectioning
  • Understanding of fixation techniques

Health and Safety (H&S)

  • School of Medical Sciences, Health and Safety Consultation 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
  • A Health and Safety Management System 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 HSMS.
  • UNSW UNSW Health and Safety Management System | Policies


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 SDS pages may be very similar, international sites may not conform to Australian Worksafe format.

Exercise



Links: Safe Work Australia - GHS | PDF | 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

Techniques for tissue and cell fixation.

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

Fresh Frozen

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.jpg
 ‎‎Cryosectioning
Page | Play


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
  • Permeabilises cells
  • Immunochemistry - some epitopes are very sensitive to methanol as it can disrupt epitope structure. (alternative is acetone for permeabilization if required).

PubChem - Methanol

Acetone

  • will also permeabilize.
  • -20C, 4C or RT.
  • fresh acetone is better, as open acetone absorbs water and increases backgrounds.


PubChem - Acetone

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%

Pubchem - Chloroform

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
  • preferred technique for many uses: immunochemistry, in situ hybridization, cell staining
  • Immunochemistry - fixation for more than 10-15 min will cross-link the proteins to the point where antigen retrieval may be required.

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.
  • Detergent's charge (nonionic, anionic, cationic, zwitterionic or amphoteric) is determined by the charge of the emulsifier or surfactant.
  • The 2 major detergent classes
    • ionic detergents (anionic, cationic)
    • nonionic detergents (uncharged, hydrophilic head groups) Tween, Triton, and the Brij series

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

Histology Stains

You would have previously covered Histology stains in your Histology course. The information below and on the linked page is provided for revision purposes.

Haematoxylin

  • UK - Haematoxylin, USA - Hematoxylin
  • Stains nuclei blue to dark-blue.
  • Stains the matrix of hyaline cartilage, myxomatous, and mucoid material pale blue.
  • Stains myelin weakly but is not noticeable if combined with eosin stain.

Eosin

  • Stains cytoplasm pink to red; red blood cells are also bright red.
  • Common counterstain to hematoxylin.
  • Stain intensity varies with the formula as well as the fixative.

Gram Stain

Gram-positive Lactobacillus rods among squamous epithelial cells and neutrophils in vaginal smear (Image CDC)

A bacterial staining procedure using crystal violet and pink safranin counterstain that generally divides bacteria into either gram-positive or gram-negative and useful for considering associated pharmacology. The procedure was named after Hans Christian Gram (1853 - 1938).

Gram-positive bacteria

  • Purple crystal violet stain is trapped by layer of peptidoglycan (peptidoglycan forms outer layer of the cell).

Gram-negative bacteria

  • Outer membrane prevents stain from reaching peptidoglycan layer in the periplasm.
    • outer membrane is composed of four major components: lipopolysaccharide, phospholipids, beta-barrel proteins, and lipoproteins.
  • outer membrane then permeabilized.
  • Pink safranin counterstain is trapped by peptidoglycan layer.


Links: Histology stains | Medical Microbiology - Gram stain procedure

Lab 3 Individual Assessment

  1. Select 4 reference papers related to your selected topic sub-section. Read these papers and write a brief description of their findings and relevance to the selected topic sub-section. The reference along with your description should then be pasted on both your group discussion page and your own personal page.
  2. Select an image related to your selected topic sub-section (this can be from one of the 4 above or from elsewhere). The image should be uploaded (with all the required information: description, reference, copyright and student template) and pasted onto the project page sub-section and onto your own personal page.


Test page

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

External Links Notice - The dynamic nature of the internet may mean that some of these listed links may no longer function. If the link no longer works search the web with the link text or name.

Cell Biology Applications of Fluorescence Microscopy Stephen Rogers Histotechniques


2015 Course Content

Lectures: Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cytoskeleton Introduction | Cytoskeleton - Microfilaments | Cytoskeleton - Microtubules | Cytoskeleton - Intermediate Filaments | Cell Mitochondria | Cell Junctions | 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 | 2015 Revision


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

2015 Projects: Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 | Group 7

Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G