ABSTRACT
Section 1. General Information Table 17.1 Solutions Commonly Used for Tissue Fixation Table 17.2 Stains Commonly Used in Histopathology Table 17.3 Common Abbreviations and Codes Used in Histopathology Table 17.4 Frequently Used Grading Schemes for Histopathology Table 17.5 Goals for Protocol Tissue Availability During Histopathology
Section 2. Common Microscopic Postmortem Changes in Laboratory Rats Section 3. Histopathologic Findings in Control Laboratory Mice Section 4. Histopathologic Findings in Control Laboratory Rats Section 5. Gross and Histopathological Findings in Control Laboratory Dogs References
Fixative Solution Primary Chemical(s) Use Tissue Recommended
(Time) Advantages/Disadvantages Comments
Formalin-saline (10%) Formaldehyde (37-40% gas in water)
Sodium chloride
General fixative for light microscopy
All (1-3 days) CNS (1-4 wk)
Easily used, rapid penetration, formic acid formation
Specimen <5 mm thickness, volume of tissue to fixative 1:>10
Move to 70% alcohol Neutral buffered formalin
(10%) Formaldehyde (37%) Sodium phosphate
buffers
General fixative for light microscopy
All (1-7 days) CNS (1-4 wk)
Easily used, rapid penetration, longer storage without formic acid formation
Specimen <5 mm thickness, volume of tissue to fixative 1:>10
Move to 70% alcohol Formalin ammonium
bromide 15% Formalin Ammonium bromide
Special fixative for nervous system
CNS (3-30 days) Preferred for Cajal gold stain for astrocytes
Bouin’s Picric acid Formaldehyde (37%) Acetic acid (15:5:1)
Brilliant colors, sharp nuclear details
Ovaries, testis, thyroid, adrenal (4-18 h)
Rapid fixation, must be replaced with 70% alcohol
Specimens thin, collect within minutes of death
Zenker’s Mercuric chloride Potassium dichromate Sodium sulfate
Brilliant colors, sharp nuclear details
Eye (6-18 h) Rapid fixation, less shinkage, poor penetration
Collect within minutes of death
Zenker acetic formalin Zenker base Formaldehyde (37%) Acetic acid
Brilliant colors, sharp nuclear details
Bone with marrow (6-18 h) Good for hematopoietic tissues Specimens thin
Carnoy’s Absolute alcohol Chloroform Acetic acid
Sharp nuclear details All (1-3 h) Rapid penetration, quick fixation, lysis of erythrocytes
Small specimens Transfer to alcohol
Helley’s (Zenker’s formalin)
Zenker base Formaldehyde (37%)
Brilliant colors, sharp nuclear details
Bone marrow (6-18 h) Good for hematopoietic tissues Specimens thin
Glutaraldehyde (2-6%) Glutaraldehyde (25%) Phosphate buffers
General fixative for electron microscopy
All (6-24 h) Rapid fixation, used for perfusion fixation of deep tissues, e.g., CNS
Secondary fixation: Osmium tetroxide, specimens <1 mm3, collect within minutes of death or perfuse
Karnovsky’s Paraformaldehyde Glutaraldehyde (50%) Phosphate buffers
General fixative for electron microscopy
All (1-2 h) Rapid fixation, used for perfusion fixation of deep tissues; e.g., CNS
Specimens <1 mm3 collect within minutes of death or perfuse, keep fixative 4° C, store in sucrose buffer
Stains Commonly Used in Histopathology1-4
Procedure Dye(s) Use or Structures Stained Result
Hematoxylin and eosin (H & E) Hematoxylin, eosin or phloxine-eosin General light microscopy Nucleus: blue Cytoplasm: red
Red blood cell: red Masson trichrome Iron hematoxylin, acid fuchsin, Ponceau 2R,
light green
General light microscopy Nucleus: black Cytoplasm: red Collagen: green Reticular fibers: green
Mallory’s phosphotungstic acid hematoxylin (PTAH)
Hematoxylin, phosphotungstic acid General light microscopy Nucleus: blue Muscle fibers: blue Collagen: red Fibrin: blue
Verhoeff’s Van Gieson Alcoholic hematoxylin, ferric chloride, Verhoeff iodine
Elastic fibers Elastic fibers: black Collagen: red Nucleus: blue Background: yellow
Weigert’s elastic Resorcin-basic fuchsin, iron hematoxylin, Van Gieson’s solution, (acid fuchsin, picric acid)
Elastic fibers Nucleus: blue Cytoplasm: yellow Collagen: red Elastic fibers: black
Gomori’s reticulum Ammoniacal silver, ferric ammonia sulfate, gold chloride
Reticulum fibers Reticulum fibers: black Background: gray
Wilder’s reticulum Ammoniacal silver, gold chloride, Mayer’s Hematoxylin
Reticulum fibers Reticulum fibers: black Collagen: red Nucleus: blue
McManus periodic acid-Schiff (PAS) with and without diastaste digestion
Periodic acid Schiff’s reagent, light green or hematoxylin, either Harris’s or Mayer’s
Carbohydrates: glycogen, glycoproteins, glycolipids in cytoplasm, basement membrane, or capsule of fungi
Glycogen: rose to purple Mucin: blue Basement membrane: pink
TABLE 17.2 (Continued) Stains Commonly Used in Histopathology1-4
Procedure Dye(s) Use or Structures Stained Result
PAS-Alcian blue Periodic acid Schiff’s reagent alcian blue, pH 2.5 or pH 1.0
Acid mucopolysaccharides, glycosaminoglycans in mucus and cartilage
Polysaccharides: red Mucosubstances: red Acid mucopolysaccharides: blue
PAS-Hematoxylin Periodic acid Schiff ’s reagent, Harris hematoxylin, light green
Carbohydrates: glycogen, glycoproteins, glycolipids in cytoplasm, basement membrane, or capsule of fungi
Glycogen: rose to purple Mucin: blue Basement membrane: pink
PAS-MS Periodic acid Schiff ’s reagent; methenamine silver
Reticulum fibers Reticular fibers: blue
Mowry’s colloid iron Ferric chloride, colloid iron, acetic acid, ferrocyanide
Acid mucopolysaccharides in cytoplasm and mucins
Hyaluronic acid: blue Sialomucin: blue
Armed Forces Institute of Pathology: Mucosubstances
Aldehyde fuchsin Mucicarmine, Mayer’s hematoxylin
Mucosubstances in mast cells and mucins Mucosubstances in mucin
Mast cells, hyaluronic acid, sialomucin, sulfated mucosubstances
Mayer’s mucicarmine Mucin: red Nucleus: blue
May-Grunwald Giemsa Giemsa Polychromasia Mast cells: blue Nucleus: blue Cytoplasm: red to blue
Nocht Azure Eosin Azure A, eosin B Polychromasia Nucleus: blue Secretory granules: red Bacteria: blue Mast cells: blue
Tomlinson-Grocott Toluidine blue Polychromasia Nucleus: blue Cytoplasm: red to blue Mast cells: dark blue
Lipid (requires frozen sections and fixation without lipid solvents)
Sudan black B Neutral lipids and triglycerides in cytoplasmic fat
Compound lipids: blue black
Neutral lipids: red
Fat (requires frozen sections and fixation without lipid solvents)
cytoplasmic fat
Simple neutral lipids: red
Bennhold Congo red, Mayer’s hematoxylin Amyloid Amyloid: pink to red Nucleus: blue
Lillie’s amyloid Crystal violet Amyloid Amyloid: red-purple Background: blue-purple
Feulgen’s HCl, Schiff’s reagent Deoxyribonucleic acid (DNA) in nuclei Nuclear DNA: red RNA (with and without ribonuclease) Methylene blue or toluidine blue Ribonucleic acid (RNA) RNA: blue staining (which is lost after
pretreatment with ribonuclease) Kluver-Berrera Luxol fast blue, cresyl violet Nervous tissue: nerve fibers Myelin: greenish blue
Cells pink to violet Einarson Gallocyanin Nervous tissue: neurons Nissl substance: dark blue
Cytoplasm: pale blue Bodian silver Protargol, hydroquinone Nervous tissue: argyrophilic granules and
nerve fibers Argyrophilic granules: black Nerve fibers: black Nucleus: black Background: gray
Gridley’s fungus Chromic acid, Feulgen reagent, aldehyde Fuchsin, metanil yellow
Fungi Fungi: deep purple Elastic tissue: purple Mucin: purple Background: yellow
Grocott’s fungus Methenamie silver, nitrate, gold chloride, light green
Fungi Fungi: black Mucin: grey Background: pale green
Brown and Brenn Gram Crystal violet, Gram’s iodine solution, basic fuchsin
Bacteria: bacterial wall Bacteria: Gram-positive: blue Gram-negative: red
Nucleus: red Background: yellow
Ziehl-Neelsen acid fast Carbol fuchsin, acid alcohol, methylene blue Acid fast bacteria, acid fast pigment Acid fast bacteria: red Acid fast pigment: red Nucleus: blue
Stains Commonly Used in Histopathology1-4
Procedure Dye(s) Use or Structures Stained Result
Von Kossa’s calcium Silver nitrate, sodium thiosulfate (Hypo), nuclear fast red
Calcium salts, bone Calcium salts: black Nucleus: red Cytoplasm: light pink
Phosphate (acid fixatives cannot be used when identifying calcium phosphate)
Silver nitrate, hydroquinone Bone, ossification: calcification Calcium phosphate: black
Perl’s iron Potassium ferrocyanide, HCL, nuclear fast red
Iron (ferric ions) Iron (ferric ions): dark blue precipitate Nucleus: red
Lead citrate Lead citrate Electron microscopy Organelle ultrastructure detailed by electron dense deposits
Uranium acetate Uranium acetate Electron microscopy Organelle ultrastructure detailed by electron dense deposits
Janigan Thioflavine-T Fluorescence microscopy: juxtaglomerular cells
Juxtaglomerular cell granules: golden yellow
Vassar-Culling Thioflavine-T Fluorescence microscopy: amyloid Amyloid: white or yellow Acridine orange fluorescence Acridine orange Fluorescence microscopy: fungi, nucleic
acids, virus particles Fungi: bright orange DNA: yellowish-green RNA: reddish-orange
Immunofluorescence Antibodies coupled to fluorescent dyes, such as fluorescein isothiocynate or rhodamin
Fluorescence microscopy: various antigenic structures
Positive antigen-antibody fluorescence
Histochemistry; acid phosphatase Glycerophosphate, lead nitrate, ammonium sulfide
Light and electron microscopy Lysosomes: black
Histochemistry; dehydrogenases Monotetrazole Light and electron microscopy Mitochondria: dark dense precipitate Histochemistry; peroxidases 3,3′ -Diaminoazobenzidine Light and electron microscopy Peroxidase sites: dense precipitate Immunohistochemistry; peroxidase Peroxidase bound to antibody, 3,3′ -
Diaminoazobenzidine Light and electron microscopy Antigen sites: dense precipitate
Immunohistochemistry; ferritin Ferritin bound to antibody Light and electron microscopy Antigen sites: dense precipitate Avidin-Biotin-Enzyme complexes Biotinylated antibody, avidin-enzyme
complex chromagen Light microscopy Antigen sites: dense or colored precipitate
TABLE 17.3 Common Abbreviations and Codes Used in Histopathology
Code Finding or Observation
+ (1) = Minimal grade lesion ++(2) = Mild or slight grade lesion +++ (3) = Moderate grade lesion ++++ (4) = Marked or severe grade lesion +++++ (5) = Very severe or massive grade lesion (No Entry) = Lesion not present or organ/tissue not examined + = Tissue examined microscopically - = Organ/tissue present, no lesion in section A = Autolysis precludes examination B = Primary benign tumor I = Incomplete section of organ/tissue or insufficient tissue for evaluation M = Primary malignant tumor M = Organ/tissue missing, not present in section N = No section of organ/tissue N = Normal, organ/tissue within normal limits NCL = No corresponding lesion for gross finding NE = Organ/tissue not examined NRL = No remarkable lesion, organ/tissue within normal limits NSL = No significant lesion, organ/tissue within normal limits P = Lesion present, not graded (for example, cyst, anomaly) R = Recut of section with organ/tissue U = Unremarkable organ/tissue, within normal limits WNL = Organ/tissues within normal limits X = Not remarkable organ/tissue, normal X = Incidence of listed morphology, lesion present
Frequently Used Grading Schemes for Histopathology5,6
Severity Degree
Proportion of Organ Affected
(A) Grade
Percentage of Organ Affected
(B) Grade
Percentage of Organ Affected
(C) Grade
Percentage of Organ Affected
(D) Grade
Percentage of Organ Affected
(E) Grade
Quantifiable Finding
Minimal Very small amount 1 (A1) < 1-25% 1 <1% 1 <1% (A2) < 1-15%
Slight Very small to small amount 2 1-25% 1 1-25% 1 1-4 foci
Mild Small amount 2 (A1) 26-50% 2 26-50% 2 1-30% 2 5-8 foci (A2) 16-35%
Moderate Middle or median 3 (A1) 51-75% 3 26-50% 3 51-76% 3 31-60% 3 9-12 foci amount (A2) 36-60%
Marked Large amount 4 (A1) 76-100% 4 >12 foci (A2) 61-100%
Moderately severe Large amount 4 51-75% Severe Very large amount 5 76-100% 4 76-100% 4 61-90% Very severe or massive Very large amount 5 91-100%
Goals for Protocol Tissue Availability During Histopathologyd 7,8
Organ or lesion Fair Range (%) >Fair = Good
Historical Availability
Fair Range (%) >Fair = Good
Historical Availability
Gross lesions <100 Unknown <100 Unknown Adrenal cortex 96-97.9 98.7 (m)a 92-95.9 97.0 (m)
98.7 (f)a 98.6 (f) Adrenal medulla 96-97.9 97.0 (m)a 92-95.9 95.9 (m)
96.1 (f)a 96.9 (f) Bone with marrow 96-97.9 97.1 (m)a 92-95.9 99.2 (m)
97.1 (f)a 99.4 (f) Brain 96-97.9 99.7 (m)a 96-97.9 99.7 (m)
99.8 (f)a 99.5 (f) Clitoral gland 92-95.9 90.3 (f)a 100.0 (f) Epididymis 92-95.9 98.3 (m)a 88-93.9 98.9 (m) Esophagus 92-95.9 95.6 (m)b 88-93.9
96.3 (f)b Gallbladder 84-91.9 87.3 (m)
90.2 (f) Heart 96-97.9 99.9 (m)a 96-97.9 99.8 (m)
99.9 (f)a 99.8 (f) Intestine, large 95.6 (m)b
96.8 (f)b Cecum 88-93.9 88-93.9 Colon 88-93.9 88-93.9 Rectum 88-93.9 88-93.9
Intestine, small 96.3 (m)b 97.8 (f)b
Duodenum 88-93.9 88-93.9 Ileum 88-93.9 88-93.9 Jejunum 88-93.9 88-93.9 Islets, Pancreas 92-95.9 98.5 (m)a 88-93.9 97.7 (m)
99.0 (f)a 97.5 (f) Kidney 96-97.9 99.7 (m)a 96-97.9 99.6 (m)
99.7 (f)a 99.4 (f) Larynx 92-95.9 88-93.9 Liver 96-97.9 99.8 (m)a 96-97.9 99.7 (m)
99.9 (f)a 99.7 (f) Lung 96-97.9 99.8 (m)a 96-97.9 99.7 (m)
99.9 (f)a 99.8 (f) Lymph node 92-95.9 88-93.9 Nasal cavity 96-97.9 97.6 (m)a 96-97.9 98.1 (m)
96.8 (f)a 98.4 (f) Ovary 96-97.9 99.6 (f)a 92-95.9 97.0 (f) Pancreas 92-95.9 98.8 (m)a 88-93.9 98.3 (m)
99.2 (f)a 98.3 (f) Parathyroid 60-79.9 89.4 (m)a 50-69.9 76.9 (m)
88.0 (f)a 77.9 (f) Pituitary gland 92-95.9 98.3 (m)a 84-91.9 91.1 (m)
99.0 (f)a 94.5 (f)
Adrenal gland, cortex Condensation of cells of zona glomerulosa (40 min*) Karyoklasis (clumping of chromatin) in epithelial cells of zona fasiculata and zona retic-
ularis (8 h) Adrenal gland, medulla
Nuclear condensation and pyknosis (2 h) Increased cytoplasmic vacuolation (2 h)
Ampullary gland Sloughed and disruption of epithelium (< 20 min)
Blood Hemolysis and pigmentation of adjacent tissues (> 16 h)
Preputial gland 92-95.9 95.4 (m)a 100.0 (m) Prostate 92-95.9 98.6 (m)a 88-93.9 97.5 (m) Salivary gland 96-97.9 97.9 (m)a 92-95.9 99.3 (m)
97.4 (f)a 98.5 (f) Seminal vesicle 92-95.9 88-93.9 Skin 96-97.9 96-97.9 Spleen 96-97.9 99.5 (m)a 92-95.9 98.9 (m)
99.7 (f)a 98.9 (f) Forestomach and stomach 92-95.9 98.8 (m)b 92-95.9 98.6 (f)b Testis 96-97.9 99.6 (m)a 92-95.9 99.4 (m) Thymus 84.9 (m)a 77.1 (m)
Chronic 60-79.9 87.5 (f)a 50-69.9 86.6 (f) Subchronic 96-97.9 92-95.9
Thyroid 94-97.9 98.9 (m)a 92-95.9 98.5 (m) 99.4 (f)a 98.5 (f)
Trachea 92-95.9 94.7 (m)b 88-93.9 95.6 (f)b
Urinary bladder 92-95.9 98.0 (m)a 92-95.9 97.6 (m) 98.4 (f)a 96.7 (f)
Uterus 92-95.9 99.1 (f)b 92-95.9 a 7142 rats, 3572 males (m), 3570 females (f). b 3919 rats, 1936 males (m), 1983 females (f). c 7596 mice, 3807 males (m), 3789 females (f). d The goal that 100% of all protocol organs and lesions will be available for histopathology is rarely possible because of sampling errors, lost tissues, and very small organs.
