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H & e staining- part 1 & 2

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H & e staining- part 1 & 2

  1. 1. Hematoxylin & Staining Presented by: 2nd Year M.D.S. Dept. of Oral & Maxillofacial Pathology & Microbiology
  2. 2. INTRODUCTION •Long history of use–Mayer 1904 •most widely used histological stain: Comparative simplicity Ability to demonstrate clearly an enormous number of different tissue structures. Hematoxylin stains cell nuclei blue black- shows good intranuclear detail. stains cell cytoplasm and most connective tissue fibers in varying shades and intensities of , orange, and red.
  3. 3. Hematoxlin - Greek word Haimato(blood) and Xylon(wood), reffering to its dark red color in natural state and to its origin(wood). HEMATOXYLIN
  4. 4. Logwood Cut end of same logwood& hematoxylin powder Hematoxylin campechianum
  5. 5. The hematoxylin is extracted from logwood with hot water, and then precipitated out fromthe aqueous solution using urea.
  6. 6. Historical Background For years it was used in textile industry until WALDEYER established its use in histology in 1862. Two years later Bohmer combined haematoxylin with alumas a mordant and obtainedmore specificstaining. Ehrlich(1886) overcame the instabilityof hematoxylin and alum by the additions of glacial acetic acidand at the same time producedhis formulafor haematoxylin as it is used today. In 1891 Heidenhain introducedhis classical Iron alum- haematoxylinmethod whichtodayis still the standard technique of the cytologist.
  7. 7. • Hematoxylin has a deep blue-purple color and stains nucleicacids by a complex, incompletely understood reaction. • Nuclei show varying cell-type- and cancer-type-specific patterns of condensation of heterochromatin(hematoxylin staining) that are diagnostically very important. • Hematoxylin, generally without eosin, is useful as a counterstain for many immunohistochemical or hybridization procedures that use colorimetric substrates (such as alkaline phosphatase or peroxidase).
  8. 8. Ripening • Hematein- Ripening • Oxidation (loss of electron) is demonstrated by the loss of hydrogen and its electron fromthe Hematoxylin structure Hematoxylin Hematein • Carried out in 2 ways – Natural oxidation Chemical oxidation
  9. 9. NATURALLYRIPENEDHEMATOXYLINS Ripening by exposure to light & air Slow process (3-4 months) Long shelf life, retain stability for a long time Example  Ehrlich’s hematoxylin  Delafield’s hematoxylin. CHEMICALLYRIPENEDHEMATOXYLINS Ripening by exposure to chemical oxidizing agents. Ripening instantaneous, ready to use immediately after preparation Shorter shelf life (because of continuing oxidation process in air & light eventually destroys much of the hematein converting it into a colourless compound) Example  Sodium iodate in Mayer’s hematoxylin (SIM)  Mercuric chloride in Harris’s hematoxylin (MCh)
  10. 10. • The process of over-oxidation of hematoxylin has established that the production of oxyhaematein inhibits successful staining- MARSHALL AND HOROBIN 1972. • Glycerol has been incorporated in many formulas as it’s a stabiliser to prevent over-oxidation and prevent evaporation. Properties of chemically oxidized hematoxylin
  11. 11. Hematein • Anionic- poor affinity for tissue - inadequate as a nuclear stain. • Mordant- A polyvalent metal ion which forms coordination complexes with certain dyes. • Net positive charge - dye-mordant complex • Type of mordant- type of tissue component and its color. • Most useful mordants - salts of aluminum, iron, and tungsten. • Lead as a mordant
  12. 12. •Biological staining – substance intermediate between dye and tissue. •Mordant forms a link between the “tissue and the stain” •Incorporation of mordant Incorporated into the hematoxylin The tissue section can be pretreated Mordant Dye LAKE
  13. 13. CLASSIFICATION I. Based on the Oxidation Procedure 1. Natural oxidation – Ehrlich’s and Delafield’s 2. Chemical Oxidation - Mayer’s and Harris II. Based on the Mordant Used 1.Alum hematoxylin 2. Iron hematoxylin 3. Tungsten hematoxylin 4. Lead hematoxylin 5. Molybedenum hematoxylin 6. Hematoxylin without mordant
  14. 14. •The mordant is aluminum in the form of a) potashalum- aluminum potassium sulfate or b) ammoniumalum-aluminum ammonium sulfate • Stains the nuclie dark red- converted to familiar blue- black- BLUING. • Specific staining: –Nuclei; DNA in chromatin • Non specific – Background staining: –Cytoplasm & Goblet cells of G.I. tract; mucin- reduced or removed by differentiation.
  15. 15. Bluing • Alkaline solutions used for bluing • Tap water is alkaline enough to produce this colour change. • Scott’s tap water substitute • Saturated Lithium carbonate (disadvantage – lithium has a tendency to form crystalline deposits unless the slides are agitated in it and well washed afterwards). • Ammonia in distilled water (disadvantage – ammonia is “hard” on delicate tissues and will loosen sections from the slide).
  16. 16. Differentiation • Provides a more controllable method in removing excess stain from tissue component and glass slide. • Traditional hcl/alcohol acts quickly and indiscriminately, is more difficult to control, and can result in light nuclear stain. • 1ml of 5 – 10% solution of acetic acid in 99ml of 70 – 95% alcohol detaches dye molecules from the cytoplasm/nucleoplasm while keeping nucleic acid complexes intact. • Exposure to air may oxidize and improve the process.
  17. 17. Regressive Staining: Stain - Regress - Continue staining (Harris hematoxylin) •Tissue is initially overstainedand then partially decolorized (differentiated) until the proper endpoint is reached. • Sharper degree of differentiation is obtained •The differentiation is controlled visually by microscopic examination. •Faster and more convenient . Progressive Staining: Stain - Rinse- Continue staining (Mayer’s, Gill’s I & II) •Tissue is stained for a predeterminedtime for adequate staining of the nuclei and leaves the backgroundtissue relatively unstained. •Once the dye is taken up by the tissues, it is not removed. •The tissue is left in the dye solution until it retains the desired amount of coloration. •The differentiationsolely relies on the selective affinity of dyes for different tissue elements.
  18. 18. Classification of Alum Hematoxylins •Ehrlich’s hematoxylin (Ehrlich 1886) •Delafield’s hematoxylin (Delafield 1885) •Mayer’s hematoxylin (Mayer 1903) •Harris hematoxylin (Harris 1900) •Cole’s hematoxylin (Cole 1943) •Carazzi’s hematoxylin (Carazzi 1991) •Gill’s hematoxylin (Gill et al 1974)
  19. 19. Ehrlich’s Hematoxylin (1886) • Naturally ripened strong alum hematoxylin. • Stains nuclei intensely and crisply • Stains mucin in salivary glands, cartilage and cement lines of bones • Suitable for tissues subjected to acid decalcification, tissues that have been stored for a long period in formalin fixatives. • Suitable for Bouin’s fixed tissue. • Not ideal for frozen sections. Ehrlich’s Hematoxylin
  20. 20. Preparation : Hematoxylin 2 g Absolute alcohol 100 ml Glycerol 100 ml Distilled water 100 ml Glacial acetic acid 10 ml Potassium alum 15 g approx. Ehrlich’s Hematoxylin
  21. 21. • The stain may be ripenednaturally by allowing to stand in a large flask, looselystoppered with cotton wool – takes about 2 months. • chemically ripened - sodium iodate - 50mg for every gram of hematoxylin. • Filter before use. Ehrlich’s Hematoxylin Dissolve the hematoxylin in alcohol Incorporate glycerol Add potassium alumtill saturation • to slow down the oxidation process and prolong shelf time.
  22. 22. • Chemically ripened with sodium iodate. • Used as both progressive and regressive stain. • Used as a nuclear counterstainin the demonstration of glycogen (PAS, mucicarmine) in various enzyme histological techniques. • Stain applied for a short period- 5-10 mins until nuclei are stained and then blued without any differentiation which might destroy/decolor the stained cytoplasmic components. Mayer’s Hematoxylin (1903) Mayer’s Hematoxylin
  23. 23. Preparation •Hematoxylin 1 g •Distilled water 1000 ml •Potassium or ammonium alum 50 g •Sodium iodate 0.2 g •Citric acid 1 g •Chloral hydrate SLR 50 g or Chloral hydrate AR 30 g Mayer’s Hematoxylin
  24. 24. • Chloral hydrate acts as a preservative • Citric acid sharpens nuclear staining boiled for 5 minutes Mayer’s Hematoxylin Hematoxylin + potassium alum + sodium iodate Chloral hydrate and citric acid cooled and filtered • dissolved in distilled water by warming & stirring
  25. 25. REFERENCES References
  26. 26. •Nuclear stains. Kiernan JA. Cold Spring Harb Protoc; doi: 10.1101/pdb. •Hematoxylin and eosin staining of tissue and cell sections. Fischer AH, Jacobson KA, Rose J, Zeller R. CSH Protoc. 2008 May 1;2008:pdb.prot4986. doi: 10.1101/pdb.prot4986 References
  27. 27. Harris’s Hematoxylin (1900) • Traditionally chemically ripened with mercuric oxide • regressive stain - routine histology practice • progressive stain - diagnostic exfoliative cytology • Differentiation is required- acetic acid alcohol. Harris’s Hematoxylin
  28. 28. Preparation •Hematoxylin 2.5 g •Absolute alcohol 25 ml •Potassium alum 50 g •Distilled water 500 ml •Mercuric oxide 1.25 g or Sodium iodate 0.5 g •Glacial acetic acid 20 ml Harris’s Hematoxylin
  29. 29. Dissolve hematoxylinin alcohol Add it to alum previously dissolvedin warmdistilledwater The mixture is rapidly broughtto boil Mercuric oxide is thenslowly and carefully added, whenthe solutionturns dark purple. The stainis rapidlycooled under tap water. Optional addition of glacial aceticacid. Filter before use. Harris’s Hematoxylin
  30. 30. Gill’s Hematoxylin (1974) •Available in 3 concentrations – o Gill’s I (normal) o Gill’s II (double) o Gill’s III (triple) most concentrated. •More frequently used than Mayer’s hematoxylin for routine H&E staining. •More stable than Harris’s hematoxylin, as auto-oxidation is inhibited to the extent. Gill’s Hematoxylin
  31. 31. Preparation of solution •Hematoxylin 2 g •Sodium iodate 0.2 g •Aluminum sulfate 17.6 g •Distilled water 750 ml •Ethylene glycol (ethandiol) 250 ml •Glacial acetic acid 20 ml Gill’s Hematoxylin
  32. 32. Advantages • Fast in action. • Stablefor at least months. • Produce little or no surface precipitate. • Their preparation doesn’t involve boiling the solution. Disadvantages • Staining of gelatin is adhesive and even the glass itself. • Some mucus may also stain darkly, as compared to Harris’s hematoxylin. Gill’s Hematoxylin
  33. 33. •Alum hematoxylin, artificially ripened with an alcoholic iodine solution. •Has good keeping qualities and is suitable for use especially in sequence with celestine blue unlike Ehrlich’s hematoxylin Cole’s haematoxylin (1943) Cole’s Hematoxylin Preparation • Hematoxylin 1.5 g • Saturated aqueous potassium alum 700 ml • 1% iodine in 95% alcohol 50 ml • Distilled water 250 ml
  34. 34. The hematoxylin is dissolved in warmdistilled water + iodine solution. The alumsolution is added and the boiled - then cooled quickly. The solution is ready for immediateuse, but mayneed on occasion filtering after storage. Filtered Cole’s Hematoxylin
  35. 35. Carazzi’s hematoxylin (1911) • Alum hematoxylin which is chemically ripened using potassiumiodate • Used as a progressive nuclear counterstain. • Largely confined to use with frozen sections Carazzi’s Hematoxylin Preparation •Hematoxylin 5 g •Glycerol 100 ml •Potassium alum 25 g •Distilled water 400 ml •Potassium iodate 0.1 g
  36. 36. Hematoxylin is dissolvedin the glycerol. Alumis dissolvedin most of the water overnight. Potassiumiodate is dissolved in the rest of the water withgentle warming & is then addedto the haematoxylin-alum-glycerol mixture. The alumsolution is added slowlyto the hematoxylin solution. Carazzi’s Hematoxylin The final staining solution is mixedwell and is then ready for immediate use.
  37. 37. Delafield’s Hematoxylin (1885) Preparation • Hematoxylin 4 g • 95% alcohol 125 ml • Saturated aqueous ammonium alum 400 ml (15 g/100 ml) • Glycerin 100 ml Delafield’s Hematoxylin
  38. 38. Delafield’s Hematoxylin The hematoxylin is dissolved in 25 ml of alcohol, and then addedto the alumsolution. This mixture is allowedto stand in light and air for 5 days and then filtered. Allowthe stain to stand exposedto light and air for about 3–4 months or until sufficiently dark in color. Glycerin and a further 100 ml of 95% alcohol are addedto this mixture. Filter before use.
  39. 39. Steps In Staining Procedure For Alum Hematoxylin Progressive Staining Methodfor paraffin sections 1. Bring sections to water with xylene and ethanol. 2. Place into the staining solution for 10 minutes. 3. Rinse with water. 4. Blue. 5. Wash well with water. 6. Counterstain if desired. 7. Dehydrate with ethanol, clear with xylene and mount with a resinous medium. Regressive Staining Method for paraffin sections 1. Bring sections to water with xylene and ethanol. 2. Place into the staining solution for 10 minutes. 3. Rinse well with water. 4. Differentiate with acid alcohol. 5. Rinse with water. 6. Blue. 7. Wash well with water. 8. Counterstain if desired. 9. Dehydrate with ethanol, clear with xylene and mount with a resinous medium. Staining
  40. 40. Staining times with alum hematoxylins Staining time varies according to various factors: • Type of hematoxylin used • Age of stain • Intensity of use of stain • Whether the stain is used progressively or regressively • Pre-treatment of tissues or sections • Post-treatment of sections • Personal preference • General rule Staining
  41. 41. Cole’s 20–45 min Delafield’s 15–20 min Ehrlich’s (progressive) 20–45 min Mayer’s (progressive) 10–20 min Mayer’s (regressive) 5–10min Harris’s (progressive in cytology) 4–30 s Harris’s (regressive) 5–15min Carazzi’s (progressive) 1–2 min Carazzi’s (frozen sections) 1 min Gill’s I (regressive) 5–15min
  42. 42. CELESTINE BLUE Preparation •Celestine blue B 2.5 g •Ferric ammonium sulfate 25 g •Glycerin 70 ml •Distilled water 500 ml
  43. 43. Ferric ammoniumsulphate is dissolved in colddistilledwater with stirring. The celestine blue B is addedto this solution and the mixture is boiledfor fewminutes. Filtered Glycerine is added Filter before use.
  44. 44. IRON HEMATOXYLINS • In these hematoxylins, iron salts such as ferric chloride and ferric ammonium sulfate are used both as the oxidizing agent and as mordant. • Over-oxidation of the hematoxylin is a problem with these stains. • Mordant/oxidant and hematoxylin solution are prepared separately and mixed before use. • Capable of demonstrating wider range of tissue structures compared to alum haematoxylin. • Techniques are more time consuming and needs microscopic control for accuracy.
  45. 45. •An iron hematoxylin used as a nuclear stainin techniques where acidic staining solutions are applied to the sections subsequently. •e.g VanGiesonstain • It is a useful stain, with eosin, for CNS tissues. Preparation Weigert’s Hematoxylin (1904) a) Hematoxylin solution • Hematoxylin 1 g • Absolute alcohol 100 ml This is allowed to ripen naturally for 4 weeks before use. b) Iron solution • 30% aqueous ferric chloride (anhydrous) 4 ml • Hydrochloric acid (concentrated) 1 ml • Distilled water 95 ml
  46. 46. • This iron hematoxylin uses ferric ammoniumsulfate as oxidant/mordant. • It is a cytological stain used as the differentiating fluid used regressively. • components are black or dark gray-black - removed progressively from different tissue structures at different rates using the iron alum solution. • Preparation Heidenhain’s hematoxylin (1896) a) Hematoxylinsolution • Hematoxylin 0.5 g • Absolute alcohol 10 ml • Distilled water 90 ml The hematoxylin is dissolved in the alcohol, and the water is then added. The solution is allowed to ripen naturally for 4 weeks before use. b) Ironsolution • Ferric ammonium sulfate 5 g • Distilled water 100 ml • It is important that only the clear violet crystals of ferric ammonium sulfate be used.
  47. 47. Loyez’s hematoxylin (1910) •This iron hematoxylin uses ferric ammoniumsulfate as the mordant. •Differentiation is by Weigert’s differentiator (borax and potassium ferricyanide). •Used to demonstrate myelin. •Can be applied to paraffin, frozen, or nitrocellulose sections.
  48. 48. Verhöeff’s hematoxylin (1908) •This iron hematoxylin is used to demonstrate elasticfibers after all routine fixative. •Ferric chloride is included in the hematoxylin staining solution, together with Lugol’s iodine, and 2% aqueous ferric chloride is used as the differentiator. •Coarse elastic fibres stain black, but the staining of fine fibers may be less than satisfactory. •The differentiation step is critical to the success of this method.
  49. 49. TUNGSTEN HEMATOXYLINS • Widely used tungsten hematoxylin is original Mallory phosphotungstic acid hematoxylin PTAH(Phosphotungstic acid hematoxylin Technique). • Used to demonstrate fibrin, muscle striations, cilia and glial fibres. • Myelin can also be demonstrated • Widely used as a CNS stain. Preparation PTAH solution using haematin Haematin 0.59 g Phosphotungstic acid 5g Distilled water 500ml Stain is ready to use immediately, but short-lived. PTAH Solution (KMnO4) Haematoxylin 0.59 g Phosphotungstic acid 5g Distilled water 500ml 0.25% Aqueous KMnO4 25 ml Peak staining activity after 7 days
  50. 50. Results •Muscle striations / neuroglia fibres / fibrin / amoeba – Dark Blue. •Nuclei/cilia/RBC – Blue •Myelin – Lighter blue •Collagen /Osteoid / Cartilage / Elastic fibres – Deep brownish red. •Cytoplasm – Pale pinkish brown.
  51. 51. MOLYBDENUM HEMATOXYLINS • Hematoxylin solution using molybdic acid as mordant- technique that gained any acceptance was the Thomas (1941). • Rare stain • Used in demonstration of collagen, coarse reticulin. • Also stains Argentaffin cell granules. PreparationPhosphomolybdic acid hematoxylin stain (Thomas 1941) Hematoxylin solution Hematoxylin 2.5 g Dioxane 49 ml Hydrogen peroxide 1 ml Phosphomolybdic acid solution Phosphomolybdic acid 16.5 g Distilled water 44 ml Diethylene glycol 11 ml
  52. 52. Results •Collagen and coarse reticulin violet to black •Argentaffin cells black •Nuclei pale blue •Paneth cells orange
  53. 53. LEAD HEMATOXYLINS •Used in the demonstration of the granules in the endocrine cells of the alimentary tract and other regions. •Most practical diagnostic application is in the identification of endocrine cells in some tumors. •Also used in research procedures such as in the localization of gastrin-secreting cells in stomach (Beltrami et al. 1975).
  54. 54. Hematoxylin without a mordant •Freshly prepared hematoxylin is used to demonstrate various minerals in tissue sections. •These methods are now replaced with more specific techniques.

Notas

  • A natural dye extracted from the core or heartwood of tree Haematoxylon campechianum.
  • natural extract obtained from the logs, hematoxylin is not an active dye.
    Hematoxylin is extracted and it is oxidised to haematein.
    Haematin is responsible for staining properties.
    The major oxidization product is hematein, a natural dye that is responsible for the color properties.

    In this process of oxidative conversion to haematin, hematoxylin loses hydrogen atoms & assumes a quinoid arrangement in one of its rings.
  • Natural Oxidation
    Carried out by exposure to light and air
    Slow process – takes about 3-4 months
    Resultant solutions seem to retain its staining ability for a long time.
    e.g Ehrlich’s & Delafield’s hematoxylin solutions

    Chemical Oxidation
    Achieved by addition of oxidizing agents such as mercuric oxide, sodium iodate and potassium permanganate.
    This process converts the hematoxylin  haematin almost instantaneously, so these hematoxylin solutions are ready for use after preparation.
  • In general, they have a shorter useful life than the naturally oxidized hematoxylins, probably because the continuing oxidation process in air and light eventually destroys much of the hematein, converting it to a colorless compound.
  • Haematein is anionic, having a poor affinity for tissue, and is inadequate as a nuclear stain without the presence of a mordant.

    The mordant/metal cation confers a net positive charge to the dye-mordant complex and enables it to bind to anionic tissue sites, such as nuclear chromatin.

    The type of mordant used influences strongly the type of tissue components stained and their final color.

    The most useful mordants for hematoxylin are salts of aluminum, iron, and tungsten, although hematoxylin solutions using lead as a mordant are occasionally used in the demonstration of argyrophil cells).


  • The mordant chemically combines with the dye (to form the lake)  and irrespective of whether the original dye is anionic, cationic, amphoteric or uncharged  the resultant combination ‘the lake’ is invariably basic in action.

    INCORPORATION OF MORDANT
    It can be incorporated into the hematoxylin staining solution (most common way).
    The tissue section can be pretreated with mordant before staining Heidenhain’s iron hematoxylin.
  • Alum Hematoxylin can be used progressively or regressively.
  • Naturally ripened strong alum hematoxylin.
    Stains nuclei intensely and crisply - stained sections fade much more slowly .
    Stains mucin in salivary glands, cartilage and cement lines of bones
    Suitable for tissues subjected to acid decalcification.
    Suitable for tissues that have been stored for a long period in formalin fixatives which have gradually become acidic over the storage period.
    Suitable for Bouin’s fixed tissue.

    not ideal for frozen sections.
  • Chemically ripened with sodium iodate.

    More vigorous in action than Ehrlich’s hematoxylin.

    Used as both progressive and regressive stain.

    Used as a nuclear counterstain in the demonstration of glycogen (PAS,mucicarmine) in various enzyme histological techniques.

    Stain applied for a short period - 5-10 mins until nuclei are stained and then blued without any differentiation which might destroy/decolor the stained cytoplasmic components.
  • This alum hematoxylin was traditionally chemically ripened with mercuric oxide (sodium or potassium iodate is frequently used as a substitute for oxidation).
    It gives particular clear nuclear staining.
    It is used as a regressive stain in routine histology practice.
    it is used as a progressive stain in diagnostic exfoliative cytology.
    When using it as a progressive stain, an acetic acid-alcohol rinse provides a more controllable method in removing excess stain from tissue components and the glass slide.
  • because it gives excellent and clear nuclear staining with a very short staining time.
  • It remains usable for about 6 months.
  • A naturally ripened alum hematoxylin with similar longevity to Ehrlich’s hematoxylin.

  • Expected results
    Nuclei ― blue
    Background ― as counterstain or unstained
    the only difference between these two procedures is that the regressive method includes a treatment with acid alcohol to remove excess staining. Some microscopists believe that sharper nuclear staining is obtained with regressive staining
  • Type of hematoxylin used
    e.g. Ehrlich’s hematoxylin - 20–45 minutes
    Mayer’s hematoxylin - 10–20 minutes.
    Age of stain. As the stain ages, the staining time will need to be increased.
    Intensity of use of stain. A heavily used hematoxylin will lose its staining powers more rapidly and longer staining times will be necessary.
    Whether the stain is used progressively or regressively e.g. Mayer’s hematoxylin used progressively 5–10 minutes and used regressively 10–20 minutes.
    Pre-treatment of tissues or sections. e.g. length of time in fixative or acid decalcifying solution or whether paraffin or frozen sections.
    Post-treatment of sections e.g. subsequent acid stains such as van Gieson.
    Personal preference.
    Shortened  for frozen sections
    Increased  for decalcified tissues
    Increased for those that have been stored for a long time in non buffered formalin.
  • Oxazine Dye
    Has little useful coloring property of its own
    It forms an additional strong mordant with certain hematoxylins.
    Used as a preliminary to alum hematoxylin staining.
    Resistant to the effects of acid.
    Ferric salt in the prepared celestine blue solution strengthens the bond between the nucleus and the alum hematoxylin to provide a strong nuclear stain which is reasonably resistant to acid.
  • Method
    1. Dewax sections, rehydrate through descending
    grades of alcohol and take to water.
    2. Stain in celestine blue solution for 5 minutes.
    3. Rinse in distilled water.
    4. Stain in an alum hematoxylin (e.g. Mayer’s or
    Cole’s) for 5 minutes.
    5. Wash in water until blue.
    6. Proceed with required staining technique.
  • 4.Iron alum allows the staining of nuclei, secretory granules, cilia, brush borders, centrosomes, mitochondria, the various transverse bands of striated muscle fibers, and bundles of cytoskeletal filaments.
  • –e.g. Van Gieson stain- picric acid is a constituent which have marked decolorizing action on nuclei stained with alum hematoxylin.
    This solution is filtered and added to an equal volume of the hematoxylin solution immediately before the stain is used. The mixture should be a violet-black color and must be discarded if it is brown.
  • Differentiation by acid alone leaves the nuclear chromatin as the last material to be destained.
    This solution is filtered and added to an equal volume of the hematoxylin solution immediately before the stain is used. The mixture should be a violet-black color and must be discarded if it is brown.

  • Two methods are similar to that of Loyez. The Heidenhain myelin stain (not to be confused with Heidenhain’s iron hematoxylin 10 The hematoxylins 182 and eosin PTAH solution, chemically oxidized with potassium permanganate
    Solution Hematoxylin 0.5 g, Phosphotungstic acid 10 g, Distilled water 500 ml, 0.25% aqueous potassium permanganate 25 ml is essentially the Loyez technique but judicious selection of staining time removes the need for separate differentiation. The second variant is the short Weil technique in which the mordant and dye are mixed before use, rather than used consecutively. Both these techniques are shorter than the Loyez.
  • Mallory phosphotungstic acid hematoxylin (PTAH) technique. Mallory (1897, 1900) combined hematoxylin with 1% aqueous phosphotungstic acid, the latter acting as the mordant.

    The hematoxylin is dissolved in 100 ml of the distilled water, and the phosphotungstic acid in the remaining 400 ml; the two solutions are mixed and the potassium permanganate solution is added. The stain can be used next day, but peak staining activity is not reached until after 7 days. Continuing oxidation of the hematoxylin means that this stain has a comparatively short life.
  • The phosphomolybdic acid solution is filtered and 50 ml of the filtrate is added to the hematoxylin solution. The resultant solution, which should be dark violet in color, is allowed to stand for 24 hours before use.

    Reticular fibers, reticular fibres or reticulin is a type of fiber in connective tissue composed of type III collagen secreted by reticular cells. Reticular fibers crosslink to form a fine meshwork (reticulin). This network acts as a supporting mesh in soft tissues such as liver, bone marrow, and the tissues and organs of the lymphatic system.

    An argentaffin cell is any enteroendocrine cell, a hormone secreting cell present throughout the digestive tract. Their granules contain a chemical called serotonin, which stimulates smooth muscle contractions.
  • Paneth cells, along with goblet cells, enterocytes, and enteroendocrine cells, represent the principal cell types of the epithelium of the small intestine.
  • argentaffin cell
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