Chapter 16: Principles of Staining

What effect can microwave-based fixation of tissue in formaldehyde have on immunohistochemical staining?

It may have an adverse effect.

What is the current recommendation for fixative solution and duration in immunohistochemical techniques?

A maximum of 4% neutral buffered formaldehyde solution and, for some antibodies, fixation time can be up to a maximum of 48 hours.

What is the main difference between immunohistochemical (IHC) staining techniques and conventional histological staining methods?

IHC techniques are based on antigenâ€"antibody bindings, which can be affected by inappropriate fixative selection and duration.

How can stains on the skin from H&E staining be effectively removed?

By prompt topical application of 0.5% acid alcohol, followed by rinsing with tap water.

What is the role of the active staining reagent in enzyme histochemistry?

It serves as a substrate upon which the enzymes act, and the final coloration produced is from the substrate rather than the tissue.

What are some tissue components that can be identified using histochemical staining techniques?

Chemical ions such as calcium, molecules such as bile pigments, and biopolymers such as cellulose, DNA, and specific enzymes.

What is the consequence of using hematoxylin solutions that have not been properly and sufficiently ripened?

The intensity of the staining reaction may be affected, leading to suboptimal staining results.

What is the purpose of histochemical staining?

To study various constituents of tissues through chemical reactions that permit microscopic localization of a specific tissue substance.

What is the main difference between regressive and progressive staining in H&E staining of frozen sections?

Regressive staining involves staining and then differentiating to achieve the desired intensity, while progressive staining builds up color intensity without differentiation, which can result in diffused color and obscured details.

How can old, bleached, or faded sections be re-stained?

Immerse the slide in xylene for 24 hours or gently heat until the mounting medium begins to bubble, then remove the coverslip and proceed with re-staining.

Why should the mercury be removed from tissues fixed with mercuric chloride before H&E staining?

Because mercury inhibits hematoxylin, and it should be removed using a 0.5% solution of iodine in alcohol, followed by rinsing in water.

What is the recommended alternative to absolute alcohol for sections treated with celloidin?

Sections treated with 95% alcohol may be transferred to a mixture of equal parts of chloroform, absolute alcohol, and xylene (C.A.X), then treated with xylene and mounted in Xam.

What could be the reason for the failure of staining, and how can it be remedied?

Failure of staining could be due to paraffin, fixative, or decalcifying solution not being thoroughly washed out, or the staining solution may be faulty. The remedy involves using fresh solutions and re-staining.

What is the consequence of using absolute alcohol in the final dehydration prior to clearing of stained sections treated with cellulose nitrate (celloidin)?

Cellulose nitrate (celloidin) is soluble in absolute alcohol and will be removed.

What is the best vital dye recommended for staining, according to the text?

Neutral red.

What is the recommended action if tissue sections appear fuzzy and unclear under the microscope after staining?

The slide should be placed in xylol to dissolve the adhesive and run back through the various processes up to the point where the fault was, using a fresh solution and re-staining the tissue.

Why should solutions for metallic impregnation never be exposed to sunlight?

Because ammoniacal silver solutions are potentially explosive and exposure to sunlight can trigger an explosion.

What is the purpose of vital staining?

To reveal cytological details that might not be apparent otherwise, and to show where certain chemicals or chemical reactions are taking place within cells or tissues.

What is the purpose of differentiating in 1% acid-alcohol during H&E staining?

To ensure that only the nuclei are stained, by monitoring the changes in color microscopically until the desired effect is achieved.

How long should slides be placed in 80% alcohol to harden the celloidin during collodionization?

3-5 minutes.

How does a mordant differ from an accentuator in staining?

A mordant is essential to the chemical union of the tissue and dye, while an accentuator merely accelerates the reaction without participating in it.

How long should Harris alum hematoxylin be applied during the staining process?

For 5 minutes, while Ehrlich's hematoxylin requires 15-30 minutes.

What is the purpose of differentiation in regressive staining?

Differentiation selectively removes excess stain from the tissue so that a specific substance may be distinctly stained from the surrounding tissues.

What is the difference between vital staining and intravital staining?

Vital staining selectively stains living cell constituents, while intravital staining involves injecting the dye into the body to color certain cells.

What is the first step in the procedure for routine H&E staining in paraffin embedded sections?

Clear paraffin embedded sections in the first xylene bath for 3 minutes.

What is the purpose of metallic impregnation in histological techniques?

To demonstrate specific tissue elements by producing an opaque, usually black deposit on the surface of the tissue or bacteria.

What is the difference between progressive and regressive staining?

Progressive staining stains tissue elements in a sequence without washing away the dye, while regressive staining involves over-staining and then removing excess stain to achieve the desired intensity.

What is metachromatic staining and when is it particularly employed?

Metachromatic staining uses specific dyes that stain certain substances with a color different from the dye itself, employed for staining cartilage, connective tissues, epithelial mucins, mast cell granules, and amyloid.

What is the term used by pathologists to indicate any area of abnormal tissue?

Lesion.

What is the significance of the phrase 'Sections to Water' in the context of staining?

It refers to the procedure of preparing a section for staining by removing paraffin and rehydrating it.

How is the differentiation process usually controlled?

By following exact times specified for staining or by examination under the microscope.

What is the purpose of deparaffinization in staining of paraffin sections?

Paraffin wax is poorly permeable to most staining solutions and should be removed from the section prior to staining.

What is the effect of section thickness on the density of the H&E stain?

Thicker sections take up more stain, affecting the stain density.

What is the result of leaving a section stained by a mordant dye in a differentiating agent like 1 to 2% alcohol?

All the dye will be removed.

What happens to a tissue component when it is decolorized in the staining process?

It becomes decolorized when a mordant oxidizes the dye to a soluble, colorless compound.

What is the most common staining method used in routine histology?

Hematoxylin and eosin (H&E) staining.

Why does Hematoxylin bind strongly to nuclear DNA?

Because Hematoxylin binds strongly to acids, and consequently to nuclear DNA.

Why are the colors of stains not indicative of the real color of a particular tissue?

Because a structure that appears one color using one stain may be a different color using another stain.

What can act as a differentiating agent besides acid, alkaline medium, and alcohol?

A mordant, such as iron alum.

What is a WBC differential and why is it useful?

A WBC differential is a test that detects abnormalities in the proportion of different white blood cells in the blood, useful for diagnosing diseases that alter these proportions.

What colors do the Hematoxylin and Eosin dyes stain the cell nuclei and other structures, respectively?

Hematoxylin stains cell nuclei blue, while Eosin stains other structures pink or red.

What is the role of a mordant in staining?

A mordant reacts with the stain to form an insoluble, colored precipitate on the tissue, making the staining reaction possible.

What is the purpose of collodionization of sections?

To more firmly attach sections to the slide, especially when subjected to strong alkaline or acid solutions or for tissues containing glycogen for demonstration.

What is the role of alcohol in the differential staining process?

Alcohol acts as a differentiator for both basic and acidic dyes by dissolving out the excess dye.

What is the primary purpose of Hematoxylin and Eosin (H&E) staining in tissue-based diagnosis?

To visualize tissue morphology and provide detail of tissue structure and cellular makeup for diagnosing infections, cancer, or metabolic diseases.

Why do different parts of a cell take up stains to varying degrees?

Because different parts of the cell are biochemically different.

How is differentiation achieved after applying a basic dye in differential staining?

By using an acid solution.

What can cause paraffin sections to float from the slide when deparaffinized and stained?

Paraffin ribbons containing air bubbles, torn or inadequately infiltrated sections.

What is the main reason cells are stained?

To enhance contrast and visualization of the cell or certain cellular components under a microscope.

What is the purpose of using more than one chemical stain in differential staining?

To better differentiate between various microorganisms or structures/cellular components of a single organism.

What is the purpose of staining in microscopy?

Staining makes tissue components visible in microscopic sections by interacting with a dye or staining solution.