Routine Staining MARCH2024 PDF

HDB 30703 Clinical Laboratory Histology 6.0 Routine Staining  Topic Outline 1. Recap 2. Introduction 3. General types of staining 4. Stain Classifications 5. Principle of dye molecule structure 6. Mechanisms of staining 7. Staining enhancer 8. Hematoxylin and e...

HDB 30703 Clinical Laboratory Histology 6.0 Routine Staining  Topic Outline 1. Recap 2. Introduction 3. General types of staining 4. Stain Classifications 5. Principle of dye molecule structure 6. Mechanisms of staining 7. Staining enhancer 8. Hematoxylin and eosin (H&E) 9. Section mounting (Coverslipping)  Topic Learning Outcomes At the end of this lecture session, students should be able to Explain the 2 basic principles of staining Explain 4 purposes of staining Explain 4 common types of staining Explain 2 Classifications of Stain dye Explain the general structure of dye molecules Discuss in detail the 3 mechanisms of staining Differentiate between 2 staining enhancers Explain H&E and discuss in detail its step-by step procedure Differentiate between progressive and regressive techniques of nuclear staining using hematoxylin. Explain the Criteria for acceptable mounting media  SPECIMEN COLLECTION 1 AND GROSSING 2 FIXATION 3 TISSUE PROCESSING Basic Histology 4 EMBEDDING Workflow or SOP 5 MICROTOMY 6 STAINING AND MOUNTING 7 QUALITY ASSURANCE INTERPRETATION AND 8 REPORTING  INTRODUCTION TO STAINING Staining The application of dyes or chemicals to biological tissues to enhance their visibility and distinguish different structures under a microscope. Principle Affinity: Dyes or stains selectively bind to specific cellular components based on their chemical properties. Contrast Enhancement: Staining differentially coloring various cellular elements, allowing easier identification and analysis.  Visualization of Structures Identification and Classification PURPOSE OF Pathological Analysis Research and Education STAINING COMMON TYPES OF STAINING Routine stain: Hematoxylin and Eosin (H&E) is widely used for general tissue morphology Staining nuclei blue-purple (hematoxylin) and cytoplasm or extracellular components pink or red (eosin). Special Stains: Target specific components like connective tissue, carbohydrates, lipids, or certain cell types. Examples include Periodic acid-Schiff (PAS) for carbohydrates or Oil Red O for lipids. Enzyme histochemical stain: Visualize and locate specific enzymes within tissues. This technique using substrates that react with the enzyme of interest to produce a visible and often colored reaction product. Immunohistochemistry (IHC): This staining technique uses antibodies to target specific proteins in tissues, aiding in the identification of specific cell types or biomarkers. STAINING DYES PRINCIPLE OF DYE MOLECULE STRUCTURE MECHANISMS OF STAINING CHEMICAL AFFINITY (ATTRACTION CHEMICAL AFFINITY (ATTRACTION Coulombic attractions van der Waals’ forces Hydrophobic bonding DENSITY PERMEABILITY STAINING ENHANCER HEMATOXYLIN AND EOSIN (H&E)  HEMATOXYLIN AND EOSIN (H&E) Hematoxylin Eosin (counterstain) Color Blue-purple. Varying shades and intensities of pink, orange or red Affinity A positively charged basic A negatively charged dye that attracts to acidic dye that attracts to negatively charged or positively charged or acidic (basophilic) basic (eosinophilic) components in cells. components in cells. Binds to basophilic Binds to eosinophilic (acidic) structures (basic) structures Targets Nucleus Cytoplasm Other Acidic Structures Extracellular Matrix HEMATOXYLIN AND EOSIN (H&E) HEMATOXYLIN AND EOSIN (H&E) HEMATOXYLIN AND EOSIN (H&E) HEMATOXYLIN AND EOSIN (H&E) HEMATOXYLIN AND EOSIN (H&E) HEMATOXYLIN AND EOSIN (H&E) HEMATOXYLIN AND EOSIN (H&E) HEMATOXYLIN AND EOSIN (H&E) HEMATOXYLIN AND EOSIN (H&E) HEMATOXYLIN AND EOSIN (H&E) HEMATOXYLIN AND EOSIN (H&E) HEMATOXYLIN AND EOSIN (H&E) HEMATOXYLIN AND EOSIN (H&E) HEMATOXYLIN AND EOSIN (H&E) THE PROGRESSIVE AND REGRESSIVE STAINING TECHNIQUES USING HEMATOXYLIN Progressive Staining Technique Regressive Staining Technique Principle A gradual buildup of stain within the Over-staining the tissue sections tissue sections followed by a subsequent treatment to remove excess dye Procedure Tissue sections are immersed in a Tissue sections are immersed in a relatively dilute hematoxylin strong and concentrated solution for a controlled period, hematoxylin solution for a longer typically shorter. duration. Treatment Often does not require a Requires a subsequent After subsequent differentiation or differentiation or bluing step to Staining bluing step as the staining is remove excess dye and achieve gradual and controlled. optimal contrast. SECTION MOUNTING (COVERSLIPPING)  References Suvarna, S.K., Layton, C. & Bancroft, J.D. (2013). Bancroft’s Theory & Practice of Histological Techniques. 7th Edition. Elsevier Thank you for your attention Do you have any questions?