Hematoxylin is a basic dye that stains the acidic components of the cell i. Nuclear counter stain for immunohistochemical staining. Acified eosin y solution, aqueous is prepared by slowly adding up to 0. For even staining results dip slides several times before allowing them to set in the eosin for the desired time. If abundant polyribosomes are present, the cytoplasm will have a distinct blue cast. Staining by hematoxylin and eosin linkedin slideshare.
The acetic acid increases the staining intensity of eosin. Haematoxylin is not classified as a dye since the molecule possesses no chromophore. Deparaffinize and hydrate to water if sections are zenkerfixed, remove the mercuric chloride crystals with iodine and clear with sodium thiosulphate hypo mayers hematoxylin for 15 minutes wash in running tap water for 20 minutes. Alcohol 50, 70, 80, 95% reagent alcohol 50, 70, 80, 95ml deionized water 50, 30, 20, 5ml staining procedure. Hematoxylineosin stain definition of hematoxylineosin. The depth or intensity of the eosin stain can be controlled by. If eosin staining is excessive, nuclear staining may be masked. It is the most widely used stain in medical diagnosis and is often the gold standard. Included in this kit is a modified eosin that provides the benefits of a traditional alcoholic formulation with significant improvements in usability. This is the standard reference stain used in the study of histochemical tissue pathology. Hematoxylin and eosin are the principle stains used in the histopathology laboratory for the demonstration of the nucleus and the cytoplasmic inclusions. The staining time in eosin the composition and length of rinses following staining following eosin staining, slides may be rinsed in water, alcohol or a mixture of the two.
This uses a combination of two dyes, hematoxylin and eosin used for demonstration of nucleus and cytoplasmic inclusions in clinical specimens. Slides are deparaffinized and rehydrated, frozens or vibratome sections are best mounted on slides and rehydrated. Nuclei show varying celltype and cancertypespecific patterns of condensation of heterochromatin hematoxylin staining that are diagnostically very important. The oxidation product of haematoxylin is haematin, and. The science and application of hematoxylin and eosin staining. Drain successively in 70% etoh for 1 minute 90% etoh for 30 seconds. Hematoxylin and eosin stain or haematoxylin and eosin stain often abbreviated as. To the final mixture add a few drops of acetic acid 0. Air dry sections for several minutes to remove moisture. Supplementary methods hematoxylin and eosin staining. Dehydrate in alcohol 70%, 95%, 100%, 3 minutes each 10.
Haematoxylin mayers s058 haematoxylin mayers is recommended for immunohistochemical and cytochemical staining as nuclear counter stain pas staining procedure. Evaluation of hematoxylin and eosin and special stains for. When paraffin sections are mounted on slides and viewed down a microscope, they appear colourless and offer little in the way of information. Dehydrate in 95% and absolute alcohols, two changes of 2 minutes each or until excess eosin is removed. Hematoxylin and eosin staining of tissue and cell sections. Please use the form below to provide feedback related to. Apart from hematoxylin and eosin, the components in the h and e staining procedure are the xylene and graded alcohols.
Eosin y 1 g distilled water 100 ml mix to dissolve. A widely used method of staining tissues for microscopic examination. To increase dierff entiation of eosin, extend time in alcohols or use a first alcohol with a higher water content. It is the most widely used stain in medical diagnosis. Alum acts as mordant and hematoxylin containing alum stains the nucleus light blue. It may also be used for routine haematoxylin and eosin staining. 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. This protocol is applied in the routine staining of cationic and anionic tissue components in tissue sections. The solution should be standardized by staining the control slides. Prepared by roy ellis imvs division of pathology the queen elizabeth hospital woodville road, woodville, south australia 5011 novaultra special stain kits principle. Clear the staining with xylene or histoclear and mount. Xylene blot excess xylene before going into ethanol.
Put the slides in a glass chamber and wash 3x for 1minute with ddh2o to remove excess of eosin. Tissue processing and hematoxylin and eosin staining. Highdefinition hematoxylin and eosin staining in a. To increase differentiation of eosin, extend time in alcohols or use a first alcohol with a higher water content. For economic reasons, the manual technique is generally the method of choice for facilities with a low sample volume. In order to improve contrast, staining the sections with dyes imparts colour to both cells and tissues. Introduction methyl green is used commonly with brightfield microscopes to stain cell nuclei by dyeing the chromatin of. The in situ oxidation of haematoxylin is effected by the addition of a strong oxidant to the stain, in this case sodium iodate.
Hematoxylin and eosin staining was performed as previously described 15. Immunohistochemical staining was performed using the manufacturers protocol, with antihuman ccl5 raised in goat, fitc. Manual hematoxylin and eosin staining of mouse tissue. Guidelines for hematoxylin and eosin staining national society for. Fisher healthcare protocol histologicalcytological stains. Proper eosin staining will demonstrate a 3tone effect. Place slides containing paraffin sections in a slide holder glass or metal deparaffinize and rehydrate sections. This document describes a method and provides reference values for the. The hematoxylin and eosin stain kit is intended for use in histology and cytology applications. Advantages include lower evaporation rate, better color patterns, and improved surface tension to remain on tissue section. Briefly, after in turn dunk in xylene and propanol for 45 s, sections were stained with hematoxylin and eosin for 45 s and 30 s, respectively. The times in the alcohols may be adjusted to obtain the proper degree of eosin staining.
The stain has been unchanged for many years because it works well with a variety of fixatives and displays a broad range of cytoplasmic, nuclear, and extracellular. Manual hematoxylin and eosin staining of mouse tissue sections. This staining procedure has remained unchanged for over 150 years. Tap water rinse slides 34 times under running tap water. This protocol is applied in the routine staining of cationic and anionic tissue components in. Immerse sections in the filtered harris hematoxylin for 1 minute. These chemicals are used to carry out the intermediate steps of rehydration and dehydration of. Blot excess water from slide holder before going into eosin. Other studies have validated hematoxylin and eosin as a useful stain for acanthamoeba.
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