IHC

Page 1: Introduction

  • Title: IHC Assay

  • Affiliation: Immunology Department, Faculty of Medicine, IUMS

  • Date: 12/09/1403

Page 2: Immunological Methods for Protein Detection

  • Methods Include:

    • Immunohistochemistry (IHC)

    • Immunofluorescence (IF)

    • Enzyme-Linked Immunosorbent Assay (ELISA)

    • Western Blotting (WB)

    • Immunoprecipitation (IP)

    • Fluorescence Activated Cell Sorting (FACS)

  • Principle of Recognition:

    • Primary antibody binds to specific epitope on protein.

  • Principle of Detection:

    • Detection done via labeled primary or secondary antibodies (e.g., HRP for IHC and WB, fluorescent dye for IF and FACS).

Page 3: Immunohistochemistry (IHC)

  • Definition: Combines immunological, histological, and biochemical techniques for identifying specific tissue components via antigen/antibody reactions.

  • Importance: Visualizes distribution and localization of specific cellular components within cells/tissues.

Page 4: History of IHC

  • Historical Context:

    • Principle has existed since the 1930s.

    • Coons identified pneumococci in 1941 using direct fluorescence.

    • Development of indirect methods:

      • Horseradish peroxidase introduction.

      • Peroxidase anti-peroxidase technique in 1979.

      • Avidin & Biotin complex usage in the early 1980s.

Page 5: Key Features of IHC

  • Antibody Interaction:

    • Specific binding of antibodies to antigens.

    • Use of a wide range of specific antibodies.

  • Detection System:

    • Highly sensitive detection system with labeled antibodies.

    • Provides spatial localization for identifying cells and proteins.

    • Useful for identifying cellular events such as apoptosis.

Page 6: Targetable Cellular Antigens

  • Types of Antigens:

    • Cytoplasmic

    • Nuclear

    • Cell membrane

    • Lipids

    • Proteins

Page 7: Types of Immunochemistry Assays

  • Types include:

    • Cells on slides (ICC).

    • Cells grown, spun into pellets, frozen or paraffin embedded and sectioned.

  • Tissue sections methods:

    • Use of frozen or paraffin embedded tissues for analysis (IHC).

Page 8: IHC vs IF

  • Overlapping Terms:

    • Direct vs Indirect methods.

  • Advantages and Disadvantages:

    • Limited labeled primary antibodies available commercially.

    • Requires more time and may be more expensive.

    • Necessity for background staining controls with limited antibodies.

    • Wide range of labeled secondary antibodies available for combination staining.

Page 9: General IHC Protocol

  • Protocol Overview:

    • Part 1: Tissue Preparation

      • Fixation, Sectioning, Whole Mount Preparation.

    • Part 2: Pretreatment

      • Antigen retrieval, inhibition of endogenous components, blocking nonspecific sites.

    • Part 3: Staining

      • Selection based on specimen type, primary antibody, sensitivity, and processing time.

Page 10: Protocol for Paraffin Embedded Tissue

  • Steps Involved:

    • Deparaffinization: Removal of infiltrated paraffin wax.

    • Rehydration: Sequential immersion in graded alcohols and PBS.

    • Antigen Retrieval: Treat deparaffinized sections with proteases or heat buffers (low/high pH).

Page 11: Protocol for Frozen Tissue

  • Tissue Sectioning:

    • Unfixed Sections: Positive for maintaining antigens but may fall off during staining.

    • Acetone Fixed: Used for many CD antibodies; precipitates proteins but may extract lipids.

    • Paraformaldehyde Fixed: Freshly made or frozen shortly; preferred over 10% buffered formalin.

Page 12: Materials for IHC and IF

  • Types of Materials:

    • Fresh or frozen tissue sections, coverslip-grown cells, sedimented cells.

    • Paraffin embedded tissue sections.

  • Considerations:

    • Limited storage time for fresh samples.

    • Individual antigen-retrieval required for paraffin sections.

Page 13: Role of Fixation in IHC

  • Fixation Functions:

    • Stabilize morphology and architecture of tissues.

    • Disable proteolytic enzymes.

    • Strengthen samples for processing and staining.

    • Protect from contamination.

  • Fixation Methods:

    • Perfusion, immersion, freezing (cryoprotective mediums), drying (for ICC).

Page 14: Common Fixatives for Antigens

  • Fixatives and Target Antigens:

    • 4% Paraformaldehyde for low molecular weight proteins.

    • Bouin's Fixative for delicate tissues.

    • Special solutions like Zenker's or Helly for blood-forming organs.

    • Ice-Cold Acetone for large protein antigens.

Page 15: Fixation Duration

  • Fixation Duration Impact:

    • Plasma urokinase inhibitor requires 48 hours fixation versus 7 days fixation.

Page 16: Sectioning Techniques

  • Sectioning Methods:

    • Paraffin: Must process through xylenes and alcohols; antigenicity may diminish after two weeks.

    • Frozen: Better antigen survival but challenges with morphology and cutting precision.

Page 17: Deparaffinization and Rehydration

  • Importance of Deparaffinization:

    • Essential for proper staining; incomplete removal can lead to staining issues.

  • Protocol Steps:

    • Wash slides in Xylene, ethanol gradients, followed by cold tap water rinse.

    • Keep slides moist to prevent drying.

Page 18: Antigen Retrieval Techniques

  • Importance:

    • Improves visibility of many antigens by breaking protein cross-links.

  • Methods:

    • Heat-Induced Epitope Retrieval (HIER) and Proteolytic Induced Epitope Retrieval (PIER).

Page 19: HIER and PIER Techniques

  • HIER Process:

    • Application of heat with retrieval solutions (citrate, Tris-EDTA, EDTA).

  • PIER Process:

    • Enzyme digestion, but risks damaging epitopes and morphology.

Page 20: Improving Antibody Penetration

  • Need for Improvement:

    • Critical for accessing intracellular components.

  • Detergents Used:

    • Triton-X, Tween for better coverage; must avoid for membrane proteins.

    • Acetone/Methanol to precipitate proteins for accessibility.

    • Saponin to puncture cell membranes safely.

Page 21: Blocking Background Staining

  • Importance of Blocking:

    • Minimizes non-specific antibody binding and background noise.

  • Common Issues:

    • Inadequate fixation and specific antibody performance.

Page 22: Non-Specific Staining

  • Management:

    • Use blocking techniques to prevent non-specific interactions causing staining artifacts.

Page 23: Background Staining Control

  • Counter-staining Techniques:

    • Utilize haematoxylin for visual contrast.

    • Identify nuclear vs. cytoplasmic antigen staining.

Page 24: Controls in IHC

  • Types of Controls:

    • Positive Control: Tissue with known specificity.

    • Negative Control: IgG from the host species against non-biologic molecules.

Page 25: Addressing IHC Signal Issues

  • Antibody-dependent signals include:

    • Non-specific signals from primary and secondary antibodies.

    • Use isotype controls for monoclonal antibodies and preimmune serum for polyclonal antibodies.

  • Antibody-independent issues need attention.

Page 26: Role of Horseradish Peroxidase (HRP)

  • Functionality:

    • Amplifies weak signals and enhances detectability.

    • Reaction: DAB + H2O2 is converted to an insoluble brown product by HRP.

Page 27: Enzymatic Detection Methods

  • Applications of Methods:

    • Brightfield microscopy for specimen analysis, lower resolution compared to fluorescence.

    • Unlimited shelf life for labeled specimens but toxic/carcinogenic substrate reagents.

Page 28: Detection Methods Overview

  • Different Methods Include:

    • Direct method, Indirect method, PAP method, ABC methods, Two-step polymer method.

Page 29: Direct and Indirect IHC Methods

  • Comparison of Direct vs Indirect Assays:

    • Direct method involves primary conjugates, whereas indirect uses secondary conjugates.

Page 30: Peroxidase-Anti-Peroxidase Complex Method (PAP)

  • Indirect Method:

    • Uses a peroxidase anti-peroxidase complex to enhance signal.

Page 31: Avidin-Biotin Complex (ABC) Method

  • Method Overview:

    • Requires preparation prior to use, employs biotinylated secondary antibodies.

Page 32: Labeled Streptavidin-Biotin Method

  • Utilizes streptavidin-enzyme complex with biotinylated secondary antibodies and primary antibodies.

Page 33: ABC Method Process

  • Workflow:

    • Sequential addition of primary and secondary antibodies and enzymes for amplification.

Page 34: SP Method Overview

  • Describes streptavidin peroxidase conjugated method process.

Page 35: Step-Polymer Method (EnVision™)

  • Process Involves:

    • Two-step amplification with dextran backbone for increased sensitivity.

Page 36: Applications

  • Fields of Use:

    • Cancer diagnostics, differential diagnosis, treatment, and research.

Page 37: Challenges in IHC

  • Common Problems and Solutions:

    • Weak signals, antibody binding issues, and improper conditions need strategic adjustments.

Page 38: High Background Solutions

  • Address Issues:

    • Optimize antibody concentrations, improve blocking efficiency, and ensure proper incubation conditions.

Page 39: Summary

  • Overview of IHC:

    • Integration of immunology, histology, and chemistry; recognizes strengths and weaknesses.

    • Importance of planning and execution for achieving reliable data.