Histotechnology: Routine Staining Part 1

Routine Staining Part 1

Introduction to Staining

• Begins with the complete removal of paraffin in fixed sections of slides
• Dewaxing starts with heating the slides in 60°C for at least 30 minutes to soften the wax
• Slides are then immersed in a couple of changes of xylene, followed by descending concentrations of alcohol to distilled water prior to primary staining

Steps Prior to Staining

• Slide preparation involves fixation, dehydration, clearing, and impregnation/infiltration
• Manual embedding technique involves using a pop-out mold to make a tissue block from the processed tissue using paraffin wax
• Important factors in manual embedding include centering or orientation of the tissue block

Cutting and Mounting

• Familiarize yourself with the parts of the microtome, its uses, and how to use it properly during cutting
• Expected to produce tissue ribbons as the final product
• Manual method of getting individual tissue sections from the ribbon involves using a floatation bath
• Albumin is used as an adhesive to the tissue section to prevent it from washing out during the staining process

Staining Mechanism

• Staining promotes easier optical differentiation and identification of cell and tissue components
• Treating tissue or cells with a series of reagents to acquire a color, making the stained element transparent
• No particles are seen, and the stained element must be transparent to have a good slide

Principles of Histological Technique

• Alteration of contrast and color are used to distinguish tissue components
• Nuclei are acidic, anionic, or negatively charged, and stain with basic dyes (blue) to exhibit a basophilic reaction
• Cytoplasm is amphoteric, and its staining depends on the pH of the solution

Dyes/Stains

• Chemical substances used to achieve visible color contrast in the microscopic picture of a prepared tissue
• Have twin properties of a color band and the ability to bind to tissues
• All dyes are organic compounds, mostly derivatives of coal tar or benzene

Integral Components of Dyes

• Chromophores: the group on the benzene ring that confers color, altering the light resonance properties of the compound
• Auxochromes: groups responsible for the tissue to bind firmly to a given dye, possessing a net charge
• Autochrome groups: amino, hydroxyl, and carboxyl groups, which are important cationic and anionic auxochromes

Classification of Dyes

• Based on pH: acidic, basic, and neutral dyes
• Based on structure: azo, quinone, and oxazine dyes
• Based on use: histological, histochemical, and immunological dyes

Qualities of a Successful Staining

• Specificity/selectivity: the ability to stain specific parts or target parts for the pathologists to observe
• Sensitivity: the ability to catch even the smallest amount of the target component### Routine Staining Part 1

Categorization of Staining

• Based on how they are used in the lab:
  • Intravital staining: injecting dye into a living animal or person to exhibit a tumor marker
  • Supravital staining: staining living cells immediately after removal from the living body
• Based on how it is done in the lab:
  • Progressive staining: staining until the desired intensity of color is attained, then excess dye is removed selectively
  • Regressive staining: overstaining, then removing excess dye to differentiate structures

Types of Staining

• Vital staining: demonstrates cytoplasmic and nuclear structures, used for identification of specific structures
• Routine staining: used in a day-to-day basis, stains various tissue elements with little differentiation except for nucleus and cytoplasm
• Selective/special staining: has little or no affinity for tissue elements, used for identification of specific structures

Staining Methods

• Direct staining: uses simple aqueous or alcoholic solutions of dye
• Indirect staining: uses a mordant or accentuator to intensify the action of the dye
• Meta-chromatic staining: uses certain dyes to exhibit a color different from the stain itself
• Counter-staining: provides contrast and background to the tissue component in target

Differentiation and Decolorization

• Selective removal of excess stain from the tissue during regressive staining
• Differentiation is controlled by following exact times specified or by microscopic examination of the section
• Differentiation is carried out by:
  • Washing a section in a simple solution
  • Using weak acids, bases, oxidizing agents, and mordants
  • Exposure to air may oxidize and improve the process

Mordants and Accentuators

• Mordant: a substance that enhances the combination of the dye with the tissues, forming a colored "lake" tissue-mordant-dye complex insoluble in aqueous and alcoholic solvents
• Accentuator: a substance that increases the speed and selectivity of the dye, but does not participate in the staining reaction
• Examples of mordants: alum, iron, and polyvalent metal ions
• Examples of accentuators: KOH, phenol, glacial acetic acid, and aniline

Requirements in Staining

• All glassware should be thoroughly cleaned to eliminate any carry-overs
• Correct solvent should be used
• Silver and osmic acid solutions should be kept in dark bottles to prevent oxidation
• Solutions like dilute ammonia should be freshly prepared due to its volatility
• Constituents of stain dissolved should follow the formula
• Alcoholic solutions of the stain should be kept in dark stoppered bottles