Talk:Histology Stains

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Cite this page: Hill, M.A. (2020, January 21) Embryology Histology Stains. Retrieved from

Stain Templates

Use the text in the curly brackets to insert the text in the curved brackets with a link to the Histology Stains page.


BRUES CT & DUNN RC. (1946). Use of fluorescent dyes as microscopical stains. Anat. Rec. , 96, 558. PMID: 20341449


(Mucin Stain, Al. carm.) Staining of acid mucopolysaccharides in tissue sections. Dye contains a large 2:1 dye-aluminum cationic complex, which is red. the mucicarmine solution is applied to sections after staining nuclei blue with a hemalum. the red dye-metal complex is attracted to anionic sites in the tissue (mucus, cartilage matrix, etc.) but it does not displace the nuclear stain. A yellow anionic dye such as picric acid or metanil yellow may optionally follow mucicarmine, as a cytoplasmic counterstain.

Chapter 9 | Carbohydrate Histochemistry John A. Kiernan, p75-92

Mucicarmine (Mucin Stain) is intended for use in the histological visualization of acid mucopolysaccharides in tissue sections. This product is useful in distinguishing mucin negative undifferentiated squamous cell lesions from mucin positive adenocarcinomas. In addition, this product will stain the mucopolysaccharide capsule of Cryptococcus neoformans.



How Romanowsky stains work and why they remain valuable - including a proposed universal Romanowsky staining mechanism and a rational troubleshooting scheme

Biotech Histochem. 2011 Feb;86(1):36-51.

Horobin RW. Source School of Life Sciences, The University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, UK.


An introduction to the nomenclature and concept of "Romanowsky stains" is followed by a brief account of the dyes involved and especially the crucial role of azure B and of the impurity of most commercial dye lots. Technical features of standardized and traditional Romanowsky stains are outlined, e.g., number and ratio of the acidic and basic dyes used, solvent effects, staining times, and fixation effects. The peculiar advantages of Romanowsky staining are noted, namely, the polychromasia achieved in a technically simple manner with the potential for stain intensification of "the color purple." Accounts are provided of a variety of physicochemically relevant topics, namely, acidic and basic dyeing, peculiarities of acidic and basic dye mixtures, consequences of differential staining rates of different cell and tissue components and of different dyes, the chemical significance of "the color purple," the substrate selectivity for purple color formation and its intensification in situ due to a template effect, effects of resin embedding and prior fixation. Based on these physicochemical phenomena, mechanisms for the various Romanowsky staining applications are outlined including for blood, marrow and cytological smears; G-bands of chromosomes; microorganisms and other single-cell entities; and paraffin and resin tissue sections. The common factors involved in these specific mechanisms are pulled together to generate a "universal" generic mechanism for these stains. Certain generic problems of Romanowsky stains are discussed including the instability of solutions of acidic dye-basic dye mixtures, the inherent heterogeneity of polychrome methylene blue, and the resulting problems of standardization. Finally, a rational trouble-shooting scheme is appended.

PMID 21235292


Non-aqueous permanent mounting for immunofluorescence microscopy

Histochem Cell Biol. 2005 Mar;123(3):329-34. Epub 2005 Mar 15.

Espada J1, Juarranz A, Galaz S, Cañete M, Villanueva A, Pacheco M, Stockert JC.


It is generally assumed that an aqueous mounting medium is necessary for the preservation of immunofluorescent-labelled microscopical preparations and polyvinyl alcohol-based solutions (e.g. Mowiol) being the most frequently used mounting media; however, both the quality and intensity of the fluorescence signal in most immunolabelled preparations after aqueous mounting slowly diminish with time, and finally, samples become unsuitable for examination. In the present work, we describe a very simple and rapid non-aqueous mounting procedure for cultured cells and tissue sections, which preserves the fluorescent signal in an excellent way after immunodetection or use of other specific labelling methods. It is based on the current histological protocol in which, after fluorescence labelling, preparations are dehydrated in ethanol, cleared in xylene and mounted in DePeX. Using this non-aqueous mounting medium, the fluorescent signal remains high and stable, allowing a suitable and permanent preservation of labelled and counterstained microscopical preparations.

PMID 15856278


Quantitative comparison of anti-fading mounting media for confocal laser scanning microscopy

J Histochem Cytochem. 2001 Mar;49(3):305-12.

Ono M1, Murakami T, Kudo A, Isshiki M, Sawada H, Segawa A.


Fading is one of the major obstacles to reliable observation in fluorescence microscopy. Using a confocal laser scanning microscope (CLSM) coupled to a computer, we quantitatively measured fading of fluorescence to formulate an equation, evaluated the anti-fading ability of several anti-fading media, and restored the faded images to the original level according to this equation. NIH 3T3 cells were stained with fluorescein isothiocyanate (FITC)-phalloidin, mounted with several commercial and homemade anti-fade media, and observed with CLSM under repeated illumination. With any mounting medium, attenuation of fluorescence intensity at a certain pixel occurred stepwise and the decrease was proportional to the intensity of the previous scan. From these results, we formulated an equation that has three coefficients: anti-fading factor (A), indicating the ability to retard fading; fluorescent intensity at the first scan (EM(1)); and background fluorescence (B). The fluorescent intensity at a certain point following nth scan is given as EM(n) = EM(1) * A ((n-1)). This equation was available for restoring faded images to their original states, even after the image had faded to only 60% of its original intensity.

PMID 11181733

Blue Histology

Wiki Histology


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)


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

Health and Safety (OHS)

  • School of Medical Sciences, 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 HS procedures and policies in the School of Medical Sciences."
  • Australian Acts and Standards
  • An HS Management System (HSMS) 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 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


  • 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)


  • 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


  • 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


  • 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


  • 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


  • 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.