Reference: Bruce-Gregorios, J., & Faldas, M.-E. (2017).

Histopathologic Techniques (US)

Table 1

ALDEHYDE FIXATIVES
Fixative Description Other Information
Formaldehyde (Formalin) Pure stock: 40%
Working solution: 10%
Neutral Buffered Formalin (10%) Best general tissue fixative (prevent autolysis and preserves tissue and cellular Advantages: cheap, readily available, compatible with many stains,
morphology) penetrates tissue well, frozen sections can be prepared with ease

Disadvantage: cause irritation of mucosa and if unbuffered: may

reduce basophilic and eosinophilic staining of cells and may form
brown pigment granules on blood containing tissue
10% Formal-Saline 40% formaldehyde diluted with 10% sodium chloride Advantages: preserve microanatomic and cytologic details with
Recommended for CNS tissues, general post-mortem tissues for histochemical minimum shrinkage and distortion, preserves enzymes and
examinations, lipids (phospholipids) nucleoproteins, demonstrates fats and mucin
Ideal for silver impregnation staining technique Disadvantage: metachromatic reaction of amyloid is reduced
Glutaraldehyde (2%; Cold and buffered) Larger molecule than formaldehyde (slower rate of diffusion) Specimen thickness should not be more than 1mm
Fixes well at 4 deg. C.; gives best overall cytoplasmic and nuclear detail Advantages: more stable effect on tissues with firmer texture (CNS);
Not used in immunohistochemical staining as it alters protein structures preserves plasma proteins, and suitable for fixation of cellular
Has 2 Reactive aldehyde groups separated by 3 carbon atoms structures
Disadvantage: expensive, penetrates tissue slowly, makes renal
biopsy tissues brittle, reduces PAS positivity of reactive mucin
Karnovsky’s Fixative 4% Paraformaldehyde, 1% glutaraldehyde in 0.1 M Phosphate buffer; must be prepared
fresh
For electron microscopy and light microscopy (samples are embedded in resin)
ALCOHOLIC FIXATIVES
• Denatures proteins and not used routinely (causes brittleness and hardness)
• Good for cytologic smears- act quickly and give nuclear detail
• PAP smears- spray cans of alcohol fixatives
Methyl alcohol (100%) Used for bone marrow and blood smear Advantages: fixes and dehydrates at the same time
Isopropyl alcohol (95%) Used for touch preparations
Ethyl alcohol (70-100%) Used as a simple fixative Disadvantages: Strong reducing agent (not to be mixed with chromic
Used in histochemistry (nucleoproteins & nucleic acids) acid, potassium dichromate, & osmium tetroxide); lower
Time: 18-24 hours concentrations cause RBCs to lyse; causes polarization (glycogen
Preserves but does not fix glycogen granules tends to move towards the poles or end of cells)

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Reference: Bruce-Gregorios, J., & Faldas, M.-E. (2017). Histopathologic Techniques (US)

Carnoy’s Fixative Most rapid fixative; Time: 1-3 hours (used for urgent biopsy specimens for paraffin Advantages: suitable for small tissue fragments
processing w/in 5 hours)
Used to fix chromosomes Disadvantage: causes RBC hemolysis, cause excessive hardening
Preserves Nissl granules and cytoplasmic granules and shrinkage, slow penetrating for large tissues, dissolves acid-
Excellent fixative for glycogen soluble cell granules and pigments
Used to fix brain tissue for the diagnosis of rabies
Clarke’s solution Used in frozen sections and smears
Time: 3-4 hours
Alcoholic Formalin Can be used in fixing large fatty specimens (e.g. breast)
Gendre’s Fixative 95% ethanol with picric acid and glacial acetic acid Disadvantages: causes partial lysis of RBC
Good for preservation of glycogen and for micro-incineration techniques
Used to fix sputum (coagulates specimen)
Newcomer’s Fluid Used in fixing mucopolysaccharides and nuclear proteins
Time: 12-18 hours at 3 deg. C
METALLIC FIXATIVES
• Unknown mechanism of action
• Increases staining brightness and gives excellent nuclear detail
• Best for fixing hematopoietic and reticuloendothelial tissues
Mercuric Chloride Most common (saturated aqueous solution of 5-7%) Disadvantages: rapidly hardens outer layer of tissue rendering
Used as a secondary fixative incomplete penetration and fixation
Usually penetrates slowly and produces shrinkage of tissues
Tissue fixed may contain black precipitate (removed before staining; sections are
treated with 0.5% iodine solution in 70% ethanol for 5-10 minutes, decolorized for 5
minutes in 5% sodium thiosulfate and washed in running water)
Routine fixative for preservation of cell detail in tissue photography
Permits brilliant metachromatic staining
Zenker’s Solution Recommended for trichome staining Disadvantages: does not permit cutting of frozen sections; causes
Recommended for congested specimens (e.g. lung, heart, blood vessels) and gives mercuric deposits but can be removed by de-zenkerization (water->
good results with PTAH and trichome staining Lugol’s iodine -> water -> sodium thiosulfate -> water)
Zenker-Formol (Helly’s) Solution Used for fixing bone marrow, extramedullary hematopoiesis, intercalated discs of
cardiac muscles, and blood containing organs
Lillie’s B-5 Fixative 4% aqueous formaldehyde with 0.22M mercuric chloride (ensures rapid structural
stabilization) and 0.22M acetic acid (coagulation of nuclear chromatin)
For bone marrow biopsies
Time: 4-8 hours
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Reference: Bruce-Gregorios, J., & Faldas, M.-E. (2017). Histopathologic Techniques (US)

Heidenhain’s Susa Solution For fixing tumor skin biopsies

Excellent cytologic fixative
OXIDIZING AGENTS
• Includes permanganate fixatives, potassium dichromate, chromic acid, and osmium tetroxide
• Used as a secondary fixative
• Forms crosslinks that stabilize tissue structure but causes extensive denaturation
Osmium tetroxide (Osmic acid; OsO4) Powder which dissolves in water Disadvantage: expensive, can cause irritation, inhibits hematoxylin
Traditionally used in EM as a fixative and heavy metal stain and counterstaining poor
Fixes conjugated fats and lipids permanently (makes it insoluble during subsequent
treatment with alcohol and xylene)
Preserves cytoplasmic structures (e.g. Golgi bodies & mitochondria)
Fixes neurological tissues (e.g. myelin and peripheral nerves)
Flemming’s solution Has glacial acetic acid
Most common chrome-osmium acetic acid fixative
Excellent fixative for nuclear structures and permanently fixes fat
Time: 24-48 hours
Flemming’s solution without acetic acid Recommended for cytoplasmic structures (mitochondria)
Time: 24-48 hours
CHROMATE FIXATIVES
Chromic acid (1-2% aq. Solution) Preserves carbohydrates
Potassium dichromate (3% aq. Solution) Preserves mitochondria
Regaud’s (Muller’s) Fluid Demonstrates chromatin, mitochondria, mitotic figures, Golgi bodies, RBC, and colloid-
containing tissues
Time: 12-48 hours
Orth’s Fluid Recommended for study of early degenerative processes and tissue necrosis
Demonstrates Rickettsia and other bacteria
PICRIC ACID FIXATIVES
• Penetrates tissue well to react with histones and basic proteins
• Good fixative for connective tissue
Bouin’s solution Recommended for fixation of embryos and pituitary biopsies Causes RBC hemolysis and reduces amount of demonstrable ferric
Excellent fixative for glycogen demonstration iron in tissue
Gives good results with tissue that subsequently stained with trichrome (Suitable for
Mallory’s, Heidenhain’s, or Masson’s staining method)
Time: 4-18 hours
Hollande’s Solution Recommended for gastro-intestinal tract specimens and endocrine tissues
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Reference: Bruce-Gregorios, J., & Faldas, M.-E. (2017). Histopathologic Techniques (US)

Has decalcifying properties

Time: 4-18 hours
Gendre’s solution Alcoholic Bouin’s solution that improves upon ageing
Preferred fixative for tissues stained by Masson’s trichrome for collagen, elastic or
connective tissue
Brasil’s Alcoholic Picroformol Fixative Excellent fixative for glycogen
OTHER FIXATIVES
Glacial Acetic Acid Precipitate DNA- preservation of nuclei (chromosomes, chromatin materials)
Michel’s Solution Not a fixative Not suitable for transporting cells for flow cytometry or for fluorescent
Transport medium for fresh unfixed tissues that will undergo frozen section and in-situ hybridization
immunofluorescence studies

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