Special Stains



Connective tissue staining



  • Collagen

    • Main protein

    • Tensile strength 

    • Type 1: most common, dense fibers

    • Type 2: hyaline cartilage

    • Type 3: reticulin fibers, meshwork of support

    • Type 4/5: Basement membranes

  • Pathology- collagen

    • Scar tissue (fibrosis)

    • Liver cirrhosis, MI, pulmonary fibrosis, tumors

    • Genetic collagen disorders



  • Masson Trichrome

    • Differentiate collagen from muscle

    • Bouin's fixative or formalin 

  1. Mordanting: section fixed in bouin’s, if formalin fixed 1h at 56C or overnight RT

  2. Wash: remove yellow from bouin’s

  3. Nuclear staining: Weigert’s hematoxylin (uses iron as mordant, ferric chloride, iron hematoxylin more resistant to decolorization

  4. Wash

  5. Acid dye (red): Beibrich scarlet/acid fuchsin (BS/AF). Both anionic (acid), red, smaller molecular size, stains acidophilic

  6. Rinse

  7. Differentiation: Phosphomolybdic acid/phophotungstic acid (PMA/PTA), larger molecules

  8. 2nd Acid dye: aniline blue, larger molecules, binds to PMA/PTA sites

  • Expected results

    • Muscle: red

    • Collagen: blue or green

  • Other collagen methods

    • Gomori one-step trichrome

      • Same as Masson’s but both acid dyes combined

    • Van Gieson picric acid-acid fuchsin

      • One solution, both dyes

      • Counterstain for elastic methods



  • Elastic fibers

    • Sheets, lung, skin, blood vessels

  • Elastin- pathology

    • Atrophy

    • Vascular conditions 

    • Distinguish veins from arteries

    • Tumors 

  • Verhoeff van Geisin

    • Hydrogen bonding, regressive staining

    • Ferric chloride and iodine act as oxidizer to convert hematoxylin to hematein also as mordant

    • Elastin has high affinity

    • Neutral buffered formalin or zenker as fixitive

    • Control: artery 

  1. Stain with Verhoeff solution (alcoholic hematoxylin + ferric chloride + Lugoi iodine)

  2. Rinse

  3. Differentiate in 2% ferric chloride (check slides)

  4. Rinse

  5. Treat with sodium thiosulfate to remove iodine 

  6. Wash

  7. Counterstain in Van Geison solution (1% acid fuchsin + picric acid)

  8. Differentiate with 95% alcohol 

  • Expected results

    • Elastic fibers: blue/black

    • Nuclei: blue

    • Collagen: red

    • other : yellow

  • Other elastic methods

    • Gomori aldehyde fuchsin

      • Better for fine elastin

      • HCL + paraldehyde added to alcoholic fuchsin = Aldehyde fuchsin 

      • Slides only hydrated up to 70% alcohol

      • Counterstain light green

      • Elastin: blue/purple   other: green



  • Other connective tissue methods

    • Movats pentachrome

      • Demonstrates mucin, fibrin, elastin

  1. Alcian blue: stains acidic mucosubstances

  2. Iron hematoxylin: stains elastin, differentiate with FeCl3

  3. Crocein scarlet/acid fuchsin: stains muscle, cytoplasm, collagen 

  4. Alcoholic safran: stains collagen

  •  Expected results:

    • Elastin: black

    • Collagen: yellow

    • Mucin: blue

    • Muscle: red



Reticulin fiber staining

  • Argentaffin = the component being demonstrated is self-reducing

  • Argyrophill = component being demonstrated cannot produce visible reaction, external reducing agent required

  • Reticulin fibers

    • Type 3 collagen

    • Liver, spleen, lymph nodes

    • High affinity for silver



  • Argyrophil reticulin methods

  1. Oxidation

  •  Hexose sugars are contained in the reticulin proteins, adjacent glycol groups of these sugars are oxidized to form aldehydes

  •  Create deposits for sensitizer

  •  Potassium permanganate

  • Bleaching after, oxalic acid

  1. Sensitization

  •  Ferric ammonium sulfate or silver nitrate

  •  Metallic salts form compound

  1. Impregnation 

  •  Tissue treated with ammoniacal silver complex, silver replace sensitizing metal

  •  Aldehyde reduce diamine silver to metallic silver

  • Argyrophill- needs external reducing agent 

  •  Incomplete wash***

  1. Reduction

  •  formaldehyde

  •  Further reduction of silver

  •  argyrophil - hydroquinone/ light

  1. Toning

  •  Treated with gold chloride

  •  Bound silver replaced with gold

  •  More stable, color changes to black

  •  Background yellow -> grey

  1. Removal of unreduced silver

  •  Sodium thiosulfate

  •  If not removed, results in black precipitate

  1. Counterstain 

  •  Should be pale

  •  Nuclear fast red, wash well or white haze will be seen

  • Expected results 

    • Nuclei, cytoplasm, muscle: red

    • Collagen: grey

    • Reticulin: black



  • Basement membrane

    • Extracellular matrix protein in sheets

    • Type 4 collagen

    • Glomerular disease

  • Periodic Acid-methanamine silver (PAMS)

    •  Jones or Brin or PAMS

    • Demonstrates carbs in basement membranes

    • Argentaffin : no external reducing agent

    • Control: kidney

  1. Oxidation

  • Periodic acid, CHO changed to aldehydes

  1. Impregnation

Methanamine silver solution, bind and reduced to metallic at 50-60C

  1. Toning

  • Gold chloride

  1. Remove excess

  • Sodium thiosulfate

  1. Counterstain: green



  • Mast cell staining

    • Toludine blue

      • Mast cells: red-purple

      • Background: blue 



Carbohydrate and amyloid staining

  • Polysaccharides

    • Group 1- neutral Polysaccharides

      • Glucose or glucosamine(chitin)

      • Liver, skeletal muscle, cervix

      • PAS reaction!!**

      • Negative with alcian blue, iron

    • Group 2- acid mucopolysaccharides

      •  Connective tissue mucins

      •  hyaluronic acid, chondroitin sulfate, aorta, cornea

      •  All anionic (acid)

      •  PAS negative

      •  Alcian blue positive

    • Group 3- glycoproteins (mucins, mucoid)

      • neutral : egg whites, stomach mucins

      • PAS variable

    • Group 4- glycolipids

      • Cerebrosides

      • Phosphatides



  • PAS (Periodic acid-Schiff)

    • Demonstrates polysaccharides, neutral muchosubstances, and basement membranes

    • Histochemical

      • Tissue oxidized to aldehydes. Aldehydes react with schiff reagent

    • Schiff reagent 

      • Basic fuchsin is reduced with sulphur

      • Reduction changes quinoid configuration of molecule and disrupts chromophore = colorless

      • Stored in fridge but used at room temp

      • Any color tint = discard

      • Reactivity test

        • Add formaldehyde

        • purple/red = good

        • Blue = discard

      • When added to tissue, binds with aldehydes, then sulphur is removed through washing = color (red/magenta)

  • PAS method 

    • Fixation

      • 10% NBF or bouin’s suitable

      • Alcoholic fixatives best

      • Glutaraldehyde to be avoided**

    • Section thickness = 4-5mm, kidney: 1-2mm

    • Controls

      • Kidney: most sensitive

      • Glycogen: liver/cervix

      • Mucin: intestine

    • Method

  1. Oxidize: create aldehyde

  • Periodic acid (weak)

  1. Wash

  2. Schiff reagent

  3. Potassium metabisulphite (optional)

  • Removes excess schiff

  1. Prolonged wash ***

  • Remove sulphur= color

  1. Counterstain 

  • Harris hematoxylin with acetic acid

  1. Wash 

  • Glycogen streaming artifact

    • Glycogen dissolving in water, gets trapped in protein

    • Cannot be corrected once tissue is fixed

    • Appearance of waves on slide



  • PAS with diastase digestion (PAS-D)

    • Specifc for glycogen

    • Same method as PAS but duplicate slides, one normal, one treated with enzyme

    • Breaks down glycogen to show if positive reaction from glycogen

  • Best Carmine

    • Demonstrates glycogen through hydrogen bonding

    • Not as specific as PAS

    • Stained with Carmine solution, differentiated with ethyl/methyl

    • glycogen = pink

  • Mayer mucicarmine

    • Epithelial mucins also stains cryptococcus

    • Useful for adenocarcinomas

    • Mucin stains= deep rose

      • Not neutral!

    • Carmine +aluminium

    • Weigerts hematoxylin (black)

    • Other= yellow

  • Alcian Blue

    • Ionic bonding

    • Basic dye (+ charge), copper phthalocyanine gives color

    • Acid mucin stains

    • pH 2.5 : both sulfated and carboxylated stain

    • pH 1.0: only sulfated

    • Fixation: 10% NBF or Bouins

    • Control: intestine

    • pH 2.5: acetic acid

    • pH 1.0: 0.1N HCl

    • Nuclear fast red as counterstain

  • Alcian Blue with hyaluronidase

    • Differentiate connective mucin from epithelial mucin

    • Put in hyaluronidase before method

    • Control: umbilical cord

  • Alcian blue-PAS-hematoxylin

    • Differentiate neutral and acidic mucins




  • Amyloid

    • Eosinophilic material replaces healthy tissue

    • Amyloidosis

      • AL- immunoglobulin light chains

    • AB- gather in brain of alzheimers patients

  • Alkaline congo red

    • Demonstrates amyloid

    • Hydrogen bonding

    • Congo red has a high affinity for amyloid

    • Alkaline Alcoholic solution solvent increased affinity 

    • Fixation: alcohol or Carnoy

    • Section: 8-10um****!!!

    • Charged slides necessary

  • Method

  1. Nuclear stain

  • Harris hematoxylin

  1. Alkaline salt solution 

  • Alcohol + NaCl + NaOH

  1. Congo red 

  • Congo red + NaCl + alcohol 



  • Results 

    • Amyloid: pink/red

    • Elastic: pale pink

    • Nuclei: blue

    • Amyloid under polarizing light: apple green**



Nerve and Lipid Staining



  • Neurons

    • Cell body + process

    • Cytoplasm contains nissl substance (basophilic)



  • Cresyl echt violet for nissl

    • Demonstrates neurons or chromatolysis

    • RNA stains sharply with basic aniline dyes

    • Alcohol differentiates

    • 6-8um



  • Nerve fibers

    • Cortex of Alzheimer's

  • Bielschowsky-PAS

    • Demonstrates nerve fibers in Alzheimer's

    • 8-10um

    • Combo silver/schiff

      • Silver deposited in neurofibers, reduced with formalin

      • Gold chloride tone

      • Sodium thiosulphate removes unreduced silver

      • Oxidized with periodic acid

      • Schiff reacts with aldehydes from CHO on plaque 

      • Results 

        • Neurofibers: black

        • Axons: black

        • Amyloid: magenta

        • Lipofuchsin: magenta

    • Other methods

      • Thioflavin 5

        • Fluorescent dye



  • Myelin sheath

    • CNS- oligodendrocytes

    • PNS- schwann cells

    • Structural support

  • Luxol fast blue

    • Myelin sheath

    • Copper phthalocyanine basic dye, soluble in alcohol

    • Basic group replacement 

    • Overnight at 56-56C

    • Differentiated by lithium carbonate

    • 10-15um** thicker!

    • Control: spinal cord/medulla

    • Results

      • Myelin: blue

      • Nuclei: violet



  • Lipids

    • Lipofuchsin

    • Fatty changes

    • Liposarcoma

    • Neutral lipids demonstrated with frozen section, processing dissolves fat

    • Stain with oil soluble dyes

      • Lysochrome must be more soluble in lipids than solvent



  • Oil red O

    • Selective solubility

    • 10um sections with charged slides

    • Counterstain in hematoxylin that does not contain alcohol

    • Aqueous mounting media

    • Results: 

      • Fat: red

      • Nuclei: blue

  • Sudan Black B

    • Neutral lipids

    • More soluble than oil red O

    • Weakly basic

    • Made with propylene glycol

    • Nuclear fast red

    • Results:

      • Fat: blue/black

      • Nuclei: red

  • Osmium teroxide 

    • Best for small fat droplets

    • Osmium tetroxides fixes lipids, turning them black

    • Very toxic, slow

    • Differentiate in periodic acid




  • Exogenous pigments

    • Carbon 

      • Lung and lymph nodes

      • Bleach resistant

    • Asbestos

      • bifringent

    • Silica

      • Unreactive and bifringent

    • Tattoo ink

  • Endogenous pigments 

    • Hemoglobin

      • Hemorrhage 

      • Intense anionic dye

    • Hemosiderin

      • Stored iron

      • yellow/brown

      • spleen/ bone marrow

    • Bile pigment

      • Biliverdin (green) or bilirubin (yellow)

    • Melanin 

      • Bleached with potassium permanganate then oxalic acid



  • Minerals 

    • Perl’s prussian blue

      • Ferric iron

      • Potassium ferrocyanide releases hemosiderin 

      • Histochemical

      • Avoid acid as it would bleach iron

      • Nuclear fast red as counterstain

    • Turnbull Blue

      • Ferrous iron

      • Potassium ferrocyanide

      • Nuclear fast red

      • Acid washed glassware

    • Schmorl method

      • Reducing substances (melanin)

      • Histochemical

      • Mayer mucicarmine, metanii yellow

      • Results

        • Reducing substances: blue/green

        • Goblet cells, mucin: rose

        • Background: yellow

    • Fontana-masson

      • Melanin and argentaffin

      • Reduces silver to visible

    • Von Kossa

      • Calcium

      • Indirect chemical method

      • Silver salts-> silver via light (UV)= black color

      • Weak sun= yellow brown 

    • Rhodanine method

      • Demonstrate copper