Principles of Staining

Principles of Staining

• Staining involves applying dyes to sections to visualize their architectural patterns and physical characteristics.

Chemical Affinity

• Acidic cell parts have a higher affinity for basic dyes, while basic cell parts prefer acidic dyes.

3 Major Groups of Staining

Histological Staining

• Tissue components are stained through direct interaction with a dye or solution.
• Examples include microanatomic, bacterial, and specific tissue stains.

Histochemical Staining

• Histochemical staining involves chemical reactions between specific tissue substances and dyes.
• Some examples include Perl's Prussian Blue for Hemoglobin and Periodic Acid Schiff for Carbohydrates.

Immunohistochemical Staining

• This staining combines immunologic and histological methods to detect phenotypic markers microscopically.
• It involves using polyclonal and monoclonal, fluorescent-labeled or enzyme-labeled antibodies.

Methods of Staining

Direct Staining

• Direct Staining involves coloring sections using aqueous or alcoholic solutions like Eosin and Methylene Blue.

Indirect Staining

• Indirect involves using a mordant or an accentuator.
• Mordants link tissue and dye.
• Mordants combine with the dye to form a "lake".
• The dye lake then combines with the tissue, forming the Tissue-Mordant-Dye Complex.
• Examples of mordants: potassium alum with Hematoxylin and iron in Weigert's Hematoxylin.
• Accentuators accelerate the staining reaction, but do not participate in it.
• Examples include potassium hydroxide (in Loeffler's Methylene Blue) and phenol (in Carbol Thionine and Carbol fuchsin).

Progressive Staining

• Stains are applied until the desired intensity is achieved.
• The distinction of tissue detail relies on the dye's selective affinity for different cellular elements.

Regressive Staining

• Regressive staining involves overstaining the tissue, and then removing or decolorizing excess stain until the desired intensity is attained.

Differentiation or Decolorization

• This process involves removing excess stain from the tissue during regressive staining, so specific substances are stained distinctly from their surroundings.
• Washing the section in solutions like water or alcohol accomplishes this.
• If the primary stain is basic, differentiation must come from an acid stain, and vice versa.
• Alcohol differentiate both acidic and basic dyes
• Mordants can also act as differentiators.
• Iron Alum oxidizes Hematoxylin into a colorless, soluble compound.

Metachromatic Staining

• Metachromatic is by using specific dyes that differentiate particular substances by staining them with a color different from the dye.
• Alcohol causes metachromasia to be lost once the tissue is dehydrated in alcohol after staining.

Counterstaining

Cytoplasmic Stains

• Red: Eosin Y, Eosin B, Phloxine B
• Yellow: Picric acid, Acid orange G, Rose Bengal
• Green: Light green SF, Lissamine green

Nuclear Stains

• Red: Neutral Red, Safranin O, Carmine, Hematoxylin
• Blue: Methylene Blue, Toluidine Blue, Celestine Blue

Metallic Impregnation

• Involves using metallic salts reduced by the tissue to produce an opaque or black deposit on the tissue surface.

Vital Staining

• Vital staining involves in the selective staining of living cell constituents
• Structures are demonstrated via phagocytosis of the dye particle.
• Reticulo-Endothelial System - stained with Trypan Blue
• Mitochondria - stained with Janus Green
• Nucleus of a living cell - resistant to vital stains

Intravital Staining

• Dye injected into any part of the animal body.
• Lithium, Carmine, and India ink are examples

Supravital Staining

• Supravital, staining of living cells immediately after removal from a living body
• Best vital stain = Neutral Red; Mitochondria = Janus Green; 1 gram of dye in 100mL = Trypan blue solution. Caution: if used more than 1 hour, it is toxic. Nile blue, Thionine, and Toluidine are examples.

Hematoxylin and Eosin Staining Technique

• Most common technique for microanatomical studies.
• Employs regressive staining technique in paraffin-embedded tissues.
• Involves overstaining of nuclei and removing excessive color from tissue constituents through acid differentiation.
• Nuclei - blue to blue black; Karyosome - Dark Blue; Cytoplasm - Pale pink

Other Staining Techniques

Heidenhain's Iron Hematoxylin Method

• Cell nuclei, cytoplasmic inclusions, and muscle striations appear black.

Celestine Blue-Haemalum Sequence Staining

• Uses an oxazine dye as an alternative to iron hematoxylin for nuclear staining.
• Celestine blue combines with iron alum, acting as a mordant to bind Hematoxylin.
• Stains Cell nuclei blue.

Mallory's Phloxine Methylene Blue Staining

• Originally known as Eosin-Methylene Blue method.

Types of Dye

Natural Dyes

• Obtained from plants and animals, and previously used for wool or nylon.

Hematoxylin

• Derived from the heartwood of Hematoxylin campechianum (Mexican Tree).
• The active coloring agent is Hematin, from the oxidation of hematoxylin or ripening.
• Natural Ripening - exposing the extract to air or sunlight for 3-4 months.
• Artificial Ripening - Adding oxidizing agents.
• Ripened Hematoxylin needs mordants like Alum, Iron, Chromium and Copper.

Cochineal Dyes

• An old histologic dye from the female cochineal bug (Coccus Cacti).
• Treats with Alum to produce the dye Carmine
• Add Picric acid (Picrocarmine) in neuropathological studies
• Add Aluminum Chloride (Best Carmine) - for glycogen

Orcein

• Comes from vegetable dye from certain lichens which are normally colorless.
• Treatment with Ammonia and exposure to air produces blue to violet colors
• Soluble with Alkali, for Elastic fibers
• Used for exposed to ammonia and air.

Artificial/Synthetic Dyes

• Derived from hydrocarbon Benzene, known as aniline dye.

Chromophores

• Substances with definite atomic groupings, capable for visible colors

Auxochrome

• Auxiliary radical that imparts electrolytic dissociation, alters the shade of the dye.

Three Groups of Artificial/Synthetic Dyes

Acid Dye

• Coloring agent is on the acidic component.
• Basic cell structures have affinity to Acidic Stains
• Examples - Acid Fuchsin; Picric Acid;

Basic Dye

• Coloring agent is on the basic component
• Acidic structures have affinity to basic stains
• Methylene blue can be used as an indicator and a dye for bacterial staining

Neutral Dye

• Formed by combining aqueous solutions of acidic and basic dyes
• Can stain cytoplasm and Nucleus simultaneously and differentially

Common Staining Solutions

Hematoxylin

• For routine histologic studies, needs the mordant Iron and Alum

Aluminum Hematoxylin

• For progressive staining and Regressive Staining
• Aluminum salts give a blue lake

Erlich's Hematoxylin

• Regressive staining.

Harris Hematoxylin

• Good regressive stain routine nuclear staining.

Cole's Hematoxylin

• Used in sequence with Celestine Blue

Carazzi's Hematoxylin

• Ripened by potassium iodide, frozen sections

Iron Hematoxylin

• For differential and regressive staining; uses acid alcohol as differentiating agent

Weigert's Hematoxylin

• Standard iron hematoxylin (Muscle fibers and Connective Tissues)

Heidenhain's Hematoxylin

• Ferric Ammonium Sulfate is the mordant
• Cytological staining, regressive staining

Loyez Hematoxylin

• Frozen Sections

Verhoeff Hematoxylin

• Mordant used is 1% Aqueous Phosphotungstic Acid
• Progressive Stain

Lead Hematoxylin

• For granules of endocrine cells of the alimentary tract

Eosin

• Eosin differentiates connective tissues and cytoplasm
• Used as a counterstain after hematoxylin and before methylene blue

Eosin Forms

• Eosin Y - Most commonly used
• Eosin B - Or Erythrosin B
• Eosin S - Or Ethyl Eosin, alcohol soluble

Carbohydrates

• Staining for carbohydrates - Carbohydrates stored in pure form (Glycogen) or bound to substances (Mucin)
• Glycogen - a polysaccharide of glucose stored in liver, heart and skeletal muscles
• Mucin - made up of hexosamines or neutral mucopolysaccharides

Periodic Acid Schiff

• Periodic acid oxidizes the 1,2 Glycol group of polysaccharides and mucin
• It liberates the aldehydes that will color the Schiff reagent - produces the red magenta or purplish pink color

Schiff Reagent

• Essential component is basic fuchsin (Made up of 3 stains, Rosanilin, Paratosanilin and Magenta II)

Staining of Mucin

• Forms the ground substance of connective tissues

Acid Mucopolysaccharides

• Bound to sulfuric acid esters and proteins
• hyaluronic acid

Proteins, Enzymes and Nucleic Acids

• Demonstration of Proteins through Histologic methods, acid histochemical methods, enzyme histochemical methods and immunocytochemical methods.

Protein Techniques

Connective Tissue

Immunohistochemistry

• Identifies specific or highly selective cellular epitopes or antigens in frozen or paraffin embedded tissues.

Epithelial Tumor Markers