List of Stains

Mechanisms of Staining

• Ionic bonding
• Hydrogen bonding
• Histochemical
  • Use a non-coloured substance to produce a coloured, non-diffusable end-product in the tissue
• Silver Staining
  • Argentaffin metallic impregnation
  • Argyophillic metallic impregnation

Summary of Conventional Staining Techniques

Stain	Uses	Tissue Components Targeted	Mechanism of Staining (Primary Stain)	Stains Used Primary Secondary	Other Reagents	Controls
Hematoxylin & Eosin (H&E)	General tissue staining	Nuclei: blue RBCs: red/dark pink Muscle: pink Collagen: light pink		Harris Hematoxylin (nuclear) Eosin Y (cytoplasm)	Acid Alcohol Scott's tap water substitute
Periodic Acid-Schiff (PAS)	Identifying neutral polysaccharides (Group I carbohydrates) glycogen starches cellulose fungi (chitin)
Alcian Blue (AB)	Identifying acid mucopolysaccharides (Group II carbohydrates) Identifying glycoproteins (Group III carbohydrates)	pH 1.0: Sulfated mucopolysaccharides: pale blue Acid mucins (mucopolysaccharides) Sialomucins (glycoproteins) Background: pink/red	Ionic bonding
Alcian Blue + Periodic Acid-Schiff (AB PAS)	Identifying multiple different types of carbohydrate groups
Congo Red	Identifying amyloid deposits		Hydrogen bonding
Masson Trichrome	Identifying fibrotic tissue	Connective Tissue Stain Nuclei: blue-black RBCs: dark red Muscle: red Cytoplasm: red Collagen: varies by stain (often green or blue)	Porosity and ionic bonding	No primary stain. Iron hematoxylin (nuclear) Ponceau (dense connective) Light green or aniline blue (loose connective)
Gordon and Sweet's Reticulin	Identifies reticulin fibres (type III & IV collagen) Differential diagnosis of certain tumour types	Connective Tissue Stain Reticulin fibres: black Nuclei and cytoplasm: varies by counterstain	Metallic impregnation Argyophilic metallic impregnation & substitution Counterstain Ionic bonding	a Varies (light green, nuclear fast red, neutral red, etc.)
Verhoeff's Van Giesen	Connective Tissue Stain Identifies elastin fibres	Connective Tissue Stain Elastic fibres: blue-black/black Nuclei: dark blue-black Collagen: red Muscle: orange RBCs: yellow Cytoplasm: yellow	Van der Waal's Primary stain Porosity and ionic bonding Counterstain	Verhoeff's Iron Hematoxylin (elastic fibres) Van Giesen Picric acid (RBCs and cytoplasm) Acid fuchsin (collagen and muscle)
Oil Red O	Connective Tissue Stain Identifying triglycerides, lipids, and lipoproteins	Fats: red	Selective solubility
Gram	Differential Microorganism Stain Gram +/- bacteria	Microorganism Stain Gram positive: purple Gram negative: pink	Ionic bonding	Crystal violet Safranin	Lugol's iodine (mordant) Acetone alcohol (decolourizer)	Positive: appendix, any tissues containing bacteria
Ziehl-Neelsen	Differential Microorganism Stain Acid fast bacteria	Microorganism Stain Acid fast: pink Non-acid fast & background: blue/pale blue	Ionic bonding	Carbol fuchsin (basic fuchsin + alcohol + 5% phenol) Methylene blue	Acid alcohol (decolourizer)	Positive: any tissue with acid fast bacteria (e.g., M. tuberculosis)
Grocott Methenamine Silver	Microorganism Stain Fungi Protozoa, encapsulated bacteria, fungal-like bacteria	Microorganism Stain Fungi: black (with paler internal structures) Background: light green	Argentaffin metallic impregnation Induced - over-oxidizes everything except fungal walls	Methenamine silver nitrate Light green or nuclear fast red	Chromic acid (oxidizer) Sodium bisulphite (bleach) Gold chloride (toner) Sodium thiosulphate (fixative)	Positive: any tissue with fungi
Perl's Prussian Blue	Iron Pigment Stain Ferric iron (hemosiderin and ferritin) Asbestos bodies	Iron: blue Nucleus: red Cytoplasm: pink	Histochemical	Perl's solution (HCl + potassium ferrocyanide) Nuclear fast red or neutral red or safranin		Positive: spleen, bone marrow, liver
Masson Fontana	Pigment Stain Argentaffin substances Melanin Argentaffin granules Neurosecretory granules	Melanin: black Nuclei: pink/red Other tissue: pale pink/brown /colourless	Argentaffin metallic impregnation	Fontana's ammoniacal silver Nuclear fast red or light green	Gram's iodine (oxidizer) Sodium thiosulphate (bleach) Gold chloride (toner) Sodium thiosulphate (fixation)	Positive: skin (melanin), small intestine (argentaffin granules) Need 4 slides to confirm Melanin! Pos QC Neg QC Unbleached patient Bleached patient (melanin removed)
Von Kossa	Pigment Stain for Calcium Stains ions bound to calcium (phosphates, carbonates, etc.)	Mineralized bone and urates: dark brown /black Non-mineralized tissue: varies by counterstain Red (NFR)	Metallic substitution - silver impregnation	von Kossa (silver nitrate) Nuclear fast red	Light or hydroquinone (reducer) Sodium thiosulphate (fixation)	Positive: any calcified tissue (e.g., bone) Can be used as negative control for: Osteoid Urates (use LiCO3) Calcium (use HCl) Need 4 slides for Ca neg control VK VK+HCl (neg) Pos QC Neg QC
Jones Methenamine Silver	Connective Tissue Stain Glomerular basement membrane	Glomerular basement membrane: black Other tissue: varies Green (LG)	Argentaffin metallic impregnation	Methanamine silver nitrate Light green or nuclear fast red	Periodic acid (oxidizer) Gold chloride (toner) Sodium thiosulphate (fixation)	Positive: normal kidney
Toluidine Blue	Connective Tissue Stain Mast cells	Metachromatic granules: red/pink/purple Nuclei and other components: varies Blue (TB) Colourless (1% acetic acid)	Metachromasia and hydrogen bonding	Toluidine blue (0.1%)	Acetic acid dips NO alcohols	Positive: tissue with mast cells (skin, small intestines)

Summary of Silver Staining Methods

Silver staining can be done via two methods: argentaffin or argyophillic staining.

• Argentaffin staining has fewer steps as the tissue components can auto-reduce the silver and do not require sensitization.
• Argyophillic methods require a sensitizing and reducing agent.

	Argentaffin Methods*			Argyophillic Methods	Metallic Substitution
Stain	Grocott's Methenamine Silver (GMS)	Jones Methenamine Silver (JMS)	Masson Fontana (MF)	Gordon & Sweet's Reticulin (GSR)	Von Kossa (VK)
Oxidation Exposes reactive sites so they can bind with silver Suppresses other tissue components Affects specificity	Chromic acid Aldehyde groups	Periodic acid	Gram's iodine Phenolic ring
Bleaching Removes discolouration from oxidizing agent	Sodium bisulphate		Sodium thiosulphate
Sensitization Helps silver attach to target
Impregnation Impregnation reagent deposits silver	Methenamine silver	Methenamine silver	Fontana ammoniacal silver		Silver nitrate
Reduction Reduces reagent to metallic silver precipitate					Light or hydroquinone
Toning Gold exchanged with silver to remove browning in non-target tissue Improves contrast Ion exchange/metallic substitution	Gold chloride	Gold chloride	Gold chloride
Fixation Removes unreduced silver	Sodium thiosulphate (hypo)	Sodium thiosulphate (hypo)	Sodium thiosulphate (hypo)		Sodium thiosulphate (hypo)
Counterstain Provides contrast to background tissue	LG or NFR	LG or NFR	LG or NFR		LG, NFR, or van Gieson

• The bleaching step is only required when the reagents themselves are coloured or cause discolouration.
• Sensitization step is not required in argentaffin methods as silver can attach directly to the target tissue.
• Argentaffin methods involve auto-reducing tissue. The tissue reduces the silver itself, so no reduction agents are needed.

Summary of Immunohistochemistry Techniques

Technique

Gram Stain

• Staining technique: Differential staining (ionic bonding to cell wall)
• Primary Stain: Crystal violet
  • Targets: Gram positive bacteria (purple/blue)
• Counterstain: Safranin
  • Targets: Gram negative bacteria (pink/red)

Ziehl-Neelsen

• Staining technique: Differential staining/acid fastness (ionic bonding)
• Primary Stain:
  • Targets: Acid-fast bacteria (e.g., Mycobacteria)
• Counterstain:
  • Targets:

Grocott Methenamine Silver

• Staining technique:
• Primary Stain:
  • Targets:
• Counterstain:
  • Targets:

Perl's Prussian Blue

• Staining technique:
• Primary Stain:
  • Targets:
• Counterstain:
  • Targets:

Masson Fontana

• Staining technique:
• Primary Stain:
  • Targets:
• Counterstain:
  • Targets:

Von Kossa

• Staining technique:
• Primary Stain:
  • Targets:
• Counterstain:
  • Targets:

Jones Methenamine Silver

• Staining technique:
• Primary Stain:
  • Targets:
• Counterstain:
  • Targets:

Toluidine Blue

• Staining technique:
• Primary Stain:
  • Targets:
• Counterstain:
  • Targets: