Hematoxylin

Hematoxylin

1. oxidations

   • Hematein
     • it is a dye from hematoxylin oxidation
     • Properties
       • -a weak anionic purple dye -poor affinity for tissue
     • enhancement
       • 1.combine to a metallic salt/mordant:substances which as as an intermediate between tissue and dye
       • The hematein become cationic dye at low pH
       • 1.use mordants
         • hematein and mordant: bind to anionic nuclear chromatin.the phosphoric acid residue in nucleic acid
         • types
           • aluminium
             • Properties
               • different aluminum salts:a source of Al+++ -The hematein forms a complex with Al+++ -cationic at most staining conditions
           • tungsten
           • iron
         • effects
           • 1. tissue components stained 2.Final color
       • 2. other addictives
         • a.polyhydroxy alcohol
           • act as a stabilizer: to reduce over oxidation of hematein
           • increase the solubility of hematein and the hematein-Al+++complexes within the solution: reasons: hematein and hematein-Al complexes are less soluble in water, may precipitate and form a metallic sheen layer on the surface of the solution
           • Glycerin
           • Ethylene glycol**
             • used to reduce precipitation: 1.increase the stability 2.shelf-life of the Hx solution
         • b. Acids
           • added to reduce pH: 1.increase the selectivity of the stain for nuclei
           • 2.reduce non-specific background staining: Cases: anioic sites:such as those found in mucins and proteoglycans,create background by binding Hmatein-Al+++ so reduction of pH:can protonate many of these sites(carry +ve charge) Application:Grill's Hx-acetic acid, Mayer's Hx -Citric acid
   • a.Natural
     • 1. exposuring to light and air 2.slow process 3-4 months 3.staining ability retain longer e.g. Ehrlich's Hx
   • b.Chemical
     • 1. sodium iodate:Mayer's Hx 2.Mecric chloride:Harris's Hx e.g. KMnO4, iodine ,feriic salt
     • properties/effects: 1.shorter shelf life:contiuous oxidation 2.Dark precipitate form: must be removed before use

2. Differentiation

   • 2.Mordant
   • 3. Oxidizing agents
   • 1.Acid
     • rountine use in alum-Hx staining

3. common categories : with mordants

   • 1. Alum hematoxylins
     • 1.routinely used in H&E 2.The forms included: potash alum-aluminum potassium sulphate ammonium sulphate: aluminum ammonium sulphate
     • staining result 1.stained nuclei red color 2.converted to blue-black when wash in a weak alkaline solution
     • progressively
       • section is stained for a predetermined time:stain the nuclei adequately but leave the background relatively unstained
     • regressively
       • section is over-stained and differentiated followed by bluing
       • require the step of differentiation(bluing)
         • converting the initially red soluble hemalum to a final blue insoluble form
         • theory: since hamulun at Low pH contribute to the reddish color,typical of acidified alum hematoxylin solution. Bluing can occur at pH 5 and up
         • types of agents
           • 1.scott's tap water substitute 2.saturated lithium substitute 3.0.05% ammonia water
         • technical concern
           • the higher the pH can faster the rate of bluing but it may loosen cell adhesion to glass
     • typical types
       • 1.Ehrlich's Hx
         • 1. stain nuclei intensely and crisply 2.fade much more slowly than otheralum Hx
         • useful for tissue: 1.subjected to acid decalcification 2.stored for a long peroid in fomalin 3.in acid fixitive not ideal for frozen sections
       • 2.Mayer's Hx
         • 1. chemically ripen with sodium iodate 2.useful as a progressive stain: counter stain nucleic where differentiation may de-color the stained cytoplasmic component 
       • 3.Harris's Hx
         • -chemically ripen with mercuric oxide (replace by sodium or potassium iodate)
         • usage: -given clear nuclear staining:progressive stain in diagnostic exfoliative cytology -use regressively in routine histological practice
         • enhancement: GAA:gives more precise and selective staining of nuclei
       • 4.Gill's Hx
         • -double or triple conc. may be used -used for routine H&E staining -more stable than Harris's Hx
         • disadvantages: -stain gelatin adhesive,even glass -stains some mucus darkly -the mordant,aluminium sulphate may responsible for
     • disadvantages
       • 1.highly sensitivity to subsequently applied acidic staining:e.g. Van Gieson -satisfactory staining can be achieved by the use of iron-mordanted Hx(e.g. Weigert's Hx) or -combination of celestine(iron-alum) blue^^^ solution with an alum-Hx
       • principal of Celestine Blue: 1. the ferric mordanted celestine blue attaches to DNA phosphate groups 2.then celestine blue is replaced by hematein 3.leaving an acid-resistant,ferric-mordanted nuclear stain
   • 2. Iron hematoxylin
     • Disadvantage: over-oxidation may result preparation: 1.mordant/oxidant, Hx solution: prepared separately. mixed before use  and use them consecutively 2. iron salt: strong oxidizing ability-used as differentiating fluid
     • a.Weigart's Hx
     • b.Heidenhain's Hx
       • ferric ammonium sulphate act as oxidant/mordant and differentiator
       • what can be stained? 1.can demonstrate chromosomes,nuclear components, mitochondria and muscle striation. 2.intestinal protozoa
     • c.Verhoeff's Hx for elastic fibers***
       • Ferric chloride(as differentiator) and Lugol's iodine are involved in the solution
       • restriction: difficult to stain both coarse and fine fibers in a single section: why?if coarse fibers are well stained ,the finest fibers may completely decolorised; if the finest fibers are stained ,the coarse fibers are under differentiated.
       • technical concern
         • the elastic fibers should be slightly underdifferentiated: picric acid in van gieson will remove some hematoxylin staining. 
     • Loyez hematoxylin for myelin
   • Tungsten
     • -1% phosphotungstic acid acts as mordant -The Hx can be oxidized byKMnO4 enhancement: staining is more precise after treating section with acid dichromate solution and Mallory bleach