Editing Haematoxylin (section)

==Use as a histologic stain==
[[File:Eosinophilic, basophilic, chromophobic and amphophilic staining.png|thumb|180px|left|Hematoxylin staining shown as "basophilic" at top, seen with dual staining with [[H&E stain|hematoxylin and eosin (H&E)]].]]
Haematoxylin stain is commonly followed (or counterstained) with another histologic stain, [[eosin]].<ref name="Chan, 2014" /><ref name="Llewellyn, 2009" /><ref name="Bancroft and Stevens, 1982" /> When paired, this staining procedure is known as [[H&E stain]]ing, and is one of the most commonly used combinations in [[histology]].<ref name="Bancroft and Stevens, 1982" /><ref name="Smith, 2006" /><ref name="Titford, 2005" /><ref name="Dapson and Horobin, 2009" /> Haematoxylin is also a component of the [[Papanicolaou stain]] (or PAP stain) which is widely used in the study of [[cytology]] specimens, notably in the [[Pap test|PAP test]] used to detect [[cervical cancer]].<ref name="Dapson and Horobin, 2009" /><ref name="Bancroft and Stevens, 1982" />

Principally used as a [[cell nuclei|nuclear]] stain (to stain the cell nucleus), haematoxylin will also stain [[rough endoplasmic reticulum]], [[ribosomes]], [[collagen]], [[myelin]], [[elastic fibers]], and acid [[mucins]].<ref name="Chan, 2014" /> Haematoxylin alone is not an effective stain, but when [[oxidized]] to hematein, and combined with a [[mordant]], stains [[chromatin]] in [[cell nuclei]] dark blue to black.<ref name="Bancroft and Stevens, 1982" /><ref name="Titford, 2005" /><ref name="Kiernan, 2018" /><ref name="Chan, 2014" /> The colour and specificity of haematoxylin stains are controlled by the chemical nature, and amount, of the mordant used, and the [[pH]] of the staining solution, thus, a variety of haematoxylin formulations have been developed.<ref name="Bancroft and Stevens, 1982" /><ref name="Chan, 2014" /><ref name="Titford, 2009" />

===Stain formulations===
[[File:Hematoxylin solution.jpg|thumb|Hematoxylin solution for staining microscopy slides.]]
Haematoxylin stain formulations can be broadly classified based on how the haematoxylin is oxidized (or ''ripened'') and by choice of the mordant used.<ref name="Bancroft and Stevens, 1982" /> Haematoxylin stain formulations may either be natural oxidized by exposure to air and sunlight, or more commonly, especially in commercially prepared solutions,<ref name="Titford, 2005" /> chemically oxidized using [[sodium iodate]].<ref name="Bancroft and Stevens, 1982" /><ref name="Gatenby and Beams, 1950" /><ref name="Llewellyn, 2009" /> Commonly only enough oxidizer is added to convert one half of the haematoxylin to haematein, allowing the remainder to naturally oxidize during use, this extends the staining solution's useful life as more haematein is produced, while some haematein is further oxidized to oxyhaematein.<ref name="Kiernan, 2006" /><ref name="Gill, 2010" /><ref name="Llewellyn, 2009" /> Of the metallic salts used as mordants, [[aluminium]] is the most common,<ref name="Llewellyn, 2009" /> other mordants include salts of [[iron]], [[tungsten]], [[molybdenum]] and [[lead]].<ref name="Bancroft and Stevens, 1982" />

Depending on the formulation or staining technique, haematoxylin stains may be used in what is called a ''progressive'' manner, in which the length of time the tissue remains in contact with the staining solution is used to control the amount of colouration, or in a ''regressive'' manner, in which the tissue is over-stained, and excess stain is removed in a secondary step of the procedure.<ref name="Llewellyn, 2009" /><ref name="Kiernan, 2018" /><ref name="Bancroft and Stevens, 1982" /> Removal of unwanted staining, or ''differentiation'', typically involves a solution of diluted [[ethanol]] and [[hydrochloric acid]].<ref name="Llewellyn, 2009" /><ref name="Bancroft and Stevens, 1982" /><ref name="Puchtler et al., 1986" />

====Table of significant formulations====
{| class="wikitable sortable"
|-
! Formula name !! Reference !! Mordant !! Oxidation method !! Typical use
|-
| [[Paul Ehrlich|Ehrlich's]] Haematoxylin<ref name="Gatenby and Beams, 1950" /> || Ehrlich, 1886 || [[Potassium alum]] || Natural || Nuclear stain in [[H&E]]
|-
| [[Francis Delafield#Legacy|Delafield's Haematoxylin]]<ref name="Gatenby and Beams, 1950" /> || Prudden, 1855 || [[Ammonium alum]] || Natural || Nuclear stain in H&E
|-
| [[Paul Mayer (zoologist)|Mayer's]] Haematoxylin<ref name="Gatenby and Beams, 1950" /> || Mayer, 1903 || Potassium or Ammonium alum || [[Sodium iodate]] || Nuclear stain in H&E
|-
| Harris's Haematoxylin<ref name="Gatenby and Beams, 1950" /> || Harris, 1900<ref name="Harris, 1900" /> || Potassium alum || [[Mercuric oxide]] || Nuclear stain in H&E, also used in the classical versions of the [[Papanicolaou stain]]<ref name="Gill, 2013" />
|-
| Cole's Haematoxylin<ref name="Bancroft and Stevens, 1982" /> || Cole, 1943<ref name="Cole, 1943" /> || Potassium alum || [[Iodine]] || Nuclear stain in H&E
|-
| Carazzi's Haematoxylin<ref name="Bancroft and Stevens, 1982" /> || Carazzi, 1911 || Potassium alum || [[Potassium iodate]] || Nuclear stain in H&E, urgent [[biopsy]] sections
|-
| [[Karl Weigert|Weigert's]] Haematoxylin<ref name="Gatenby and Beams, 1950" /> || Weigert, 1904 || [[Ferric chloride]] || Natural || Nuclear stain in H&E, resistant to acids
|-
| [[Verhoeff's stain|Verhoeff's Haematoxylin]]<ref name="Bancroft and Stevens, 1982" /> || Verhoeff, 1908 || Ferric chloride || [[Iodine]] || [[elastic fibers]], [[myelin]]<ref name="Puchtler et al., 1986" />
|-
| [[Frank Burr Mallory|Mallory's]] [[Phosphotungstic acid haematoxylin|phosphotungstic acid Haematoxylin]]<ref name="Bancroft and Stevens, 1982" /> || Mallory, 1897 || [[Phosphotungstic acid]] || Natural or chemical || [[Fibrin]], [[Striated muscle tissue|muscle striations]]
|-
| Gill's Haematoxylin (I, II, and III)|| Culling et al. 1985 <ref name="Llewellyn, 2009" /><ref name="Gill, 2010" /> || [[Aluminium sulfate]] || Sodium iodate || Nuclear stain in H&E
|}

===Early use as a histologic stain===
In 1758, Georg Christian Reichel used haemotoxylin, without a mordant, to stain plant tissues.<ref name="Bracegirdle 1986" /><ref name="Smith, 2006" /><ref name="Allison, 1999" /> [[John Thomas Quekett]] in an 1852 book,<ref name="Quekett, 1852" /> suggests using "logwood" (haematoxylin) to dye translucent material for examination under the [[microscope]].<ref name="Allison, 1999" /><ref name="Bracegirdle 1986" /> In 1863, [[Wilhelm von Waldeyer-Hartz]] used haematoxylin on animal tissue without a mordant (with limited success),<ref name="Mann, 1902" /> and is sometimes credited as being the first to do so,<ref name="Ortiz-Hidalgo and Pina-Oviedo, 2019" /><ref name="Smith, 2006" /><ref name="Cook, 1997" /><ref name="Mann, 1902" /> although this is not universally accepted.<ref name="Cook, 1997" /><ref name="Ortiz-Hidalgo and Pina-Oviedo, 2019" /> Franz Böhmer in 1865 published a haematoxylin formula using alum as a mordant,<ref name="Mann, 1902" /><ref name="Cooksey, 2010" /><ref name="Smith, 2006" /><ref name="Ortiz-Hidalgo and Pina-Oviedo, 2019" /><ref name="Cook, 1997" /><ref name="Bracegirdle 1986" /> and in 1891, [[Paul Mayer (zoologist)|Paul Mayer]] published a formulation using a chemical oxidizer to convert haematoxylin into haematein.<ref name="Gatenby and Beams, 1950" /><ref name="Bracegirdle 1986" /><ref name="Smith, 2006" /> The first use of haematoxylin with eosin as a counterstain, which is currently the most used stain combination in histology, was first suggested by A. Wissowzky in 1876.<ref name="Titford, 2009" /><ref name="Bracegirdle 1986" /> By the early 1900s, haematoxylin had become widely accepted as a histologic stain.<ref name="Smith, 2006" />

===Shortages and possible alternatives===
During [[World War I]], the late 1920s, [[World War II]], the early 1970s (summer 1973<ref name="Lillie, 1974" />) and in 2008, there were shortages of haematoxylin due to interruptions in its extraction from logwood.<ref name="Dapson et al., 2010" /> These shortages prompted a search for alternative nuclear stains.<ref name="Lillie, 1974" /><ref name="Dapson et al., 2010" /> Several synthetic dyes have been recommended as replacements, notably celestine blue (CI 51050),<ref name="Dapson et al., 2010" /> [[Gallocyanin stain|gallocyanine]]<ref name="Titford, 2005" /><ref name="Llewellyn, 2009" /> (CI 51030), gallein<ref name="Dapson et al., 2010" /> (CI 45445) and [[Cyanine#Nucleic Acid Labeling|eriochrome cyanine R]]<ref name="Dapson et al., 2010" /><ref name="Llewellyn, 2009" /> also called chromoxane cyanine R and solochrome cyanine (CI 43820). All four have Fe(III) as the [[mordant]]. An alternative is the aluminium complex of oxidized [[brazilin]], which differs from haematoxylin by only one hydroxyl group. A replacement stain for haematoxylin in [[H&E|H&E staining]] must also not disrupt the ability of histologists and pathologists,<ref name="Dapson and Horobin, 2009" /> who have spent years of training with H&E stained slides, to examine the slides and make [[diagnoses|medical diagnoses]].<ref name="Titford, 2005" /> None of proposed replacement stains have been widely adopted.<ref name="Dapson and Horobin, 2009" /><ref name="Titford, 2005" />