Introduction

This is an introduction to histological techniques of tissue/cell fixation and should be considered in relation to Histology stains and other analytical techniques. Also consider
the Occupational Health and Safety (OHS) issues in relation to chemicals used in this process.
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 histological staining techniques

Brief understanding of tissue preparation and sectioning

Understanding of fixation techniques

Brief understanding of chemical OHS issues

Three Main Techniques

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

PubChem - Methanol

Chloroform-containing Fixative

Carnoys 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

Uses: immunochemistry, in situ hybridization, cell staining

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

Gluteraldehyde

Aldehyde Cross-Link fixation

Other Fixation Considerations

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

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.

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

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.