25 Questions
What is the purpose of using DPX or XPF on coverslip glass in the alternative dry mount method?
To prevent the staining from fading over time
What is the color of connective tissue as observed in the H&E results?
Pale pink
What is the potential impact of varying the thickness of cut sections on H&E staining variability?
Affects the balance of staining time and concentration of the nuclear stain
What is the recommended thickness for achieving an optimum balance in H&E staining?
4 micron
Which of the following is the mechanism of staining used in the direct method?
Attraction of acidic elements to basic dyes
What is the function of a mordant in the indirect method of staining?
To link the tissue and dye
What is the color of the dye lake produced when a mordant is combined with a dye?
Dependent on the specific mordant used
What is the process involved in progressive staining?
Up to the point for the desired depth of coloration to be achieved
Which tissue element has negative charges and reacts with haematoxylin?
Nuclei
What reaction occurs when haematoxylin, a basic dye, reacts with acidic nuclei?
Blue/black coloration
What color does eosin, an acid dye, produce when it reacts with basic tissue elements?
Red/shades of pink
What is the purpose of regressive staining?
Overstaining of tissue followed by removal of the stain
What is the charge of RBCs, cytoplasm, muscle, and collagen?
Basic (+ve charges)
What reaction occurs when eosin, an acid dye, reacts with RBCs, cytoplasm, muscle, and collagen?
Red/shades of pink coloration
What is the function of Haematoxylin in the H&E staining technique?
To illustrate nuclear detail in cells
Which substance is oxidized to produce hematein, the actual dye used in an H&E stain?
Haematoxylin
What is the purpose of using a mordant such as aluminum ammonium sulfate (alum) in the H&E staining process?
To improve the bonding of hematein to the anionic tissue components
What is the primary function of Eosin in the H&E staining technique?
To counterstain and distinguish between the cytoplasm and nuclei of cells
Why is filtering required for Harris hematoxylin, a strong, regressive stain?
To remove precipitates
Which substance is known for fading rapidly in sunlight, requiring labs to use Eosin Y as a counterstain?
Eosin
What step follows haematoxylin staining in the basic protocol for H&E staining?
Differentiating with acid alcohol
What is responsible for staining cytoplasm in H&E staining?
Harris hematoxylin
What solution is commonly used for differentiating Harris hematoxylin in most labs?
1% or 0.5% HCl in 70% alcohol
What step follows counterstaining with eosin in the staining schedule for H&E?
Differentiating with acid alcohol
What feature is very crisp in an example of H&E staining?
Microvilli on the columnar epithelium
Study Notes
- Hematoxylin and Eosin (H&E) is the most commonly used staining technique in histology for diagnosing malignancies.
- Haematoxylin, derived from Haematoxylon campechianum (bloodwood), is used to illustrate nuclear detail in cells, with the depth of coloration depending on the length of time the sample spends in haematoxylin.
- Haematoxylin is oxidized to produce hematein, which is the actual dye used in an H&E stain, and the use of a mordant such as aluminum ammonium sulfate (alum) improves the bonding of hematein to the anionic tissue components, most commonly chromatin.
- Eosin, a red crystalline dye, is the most commonly used counterstain that distinguishes between the cytoplasm and nuclei of cells.
- Histopathology diagnosis relies heavily on H&E staining, demonstrating a broad range of cytoplasmic, nuclear, and extracellular matrix features, and producing a crisp nuclear staining with Harris hematoxylin.
- Harris hematoxylin is a strong, regressive stain that produces well-defined nuclear outlines but requires filtering to remove precipitates, and most labs use an aggressive differentiating solution like 1% or 0.5% HCl in 70% alcohol for a few seconds.
- Eosin is not permanent and fades rapidly in sunlight, with Eosin Y being the most commonly used form of eosin.
- The staining procedure for H&E follows a basic protocol including dewaxing, rehydration, haematoxylin staining, differentiation, and counterstaining with eosin, among other steps.
- The balanced coloration of the stained specimen is the result of the intensity of the alum-hematoxylin and eosin, with alum-hematoxylin able to stain cytoplasm and eosin Y able to stain nuclear basic protein.
- The staining schedule for H&E includes various steps such as immersing the slide in xylene, washing with alcohol, staining with Harris' haematoxylin, differentiating with acid alcohol, and counterstaining with eosin, among others.
- The microvilli on the columnar epithelium are very crisp in an example of H&E staining.
Test your knowledge of the mechanism of Hematoxylin and Eosin (H&E) staining with this quiz focused on the direct method of staining, the attraction between tissue elements and dyes, and the concept of basophilic and acidophilic components.
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