Immunohistochemistry Course

Lesson 1 • Tissue Architecture and Cell Biology

Reviews cell organelles, extracellular matrix, and tissue organization. Contextualizes where target antigens reside within tissue sections.

Lesson 2 • Antigen-Antibody Interaction Basics

Covers epitope structure, antibody classes, and binding affinity. Provides the molecular rationale for every downstream IHC reagent choice.

Lesson 3 • Laboratory Safety and Regulatory Compliance

Addresses chemical hazards, biosafety levels, and waste disposal. Ensures students operate within applicable safety and quality standards.

Lesson 1 • Cryostat and Frozen Section Preparation

Introduces snap-freezing, cryostat operation, and frozen section mounting. Frozen sections preserve labile antigens lost during paraffin processing.

Lesson 2 • Fixation Chemistry and Methods

Explains aldehyde cross-linking, coagulative fixatives, and fixation kinetics. Fixative choice directly determines antigen preservation and retrieval needs.

Lesson 3 • Tissue Processing and Embedding

Teaches dehydration, clearing, infiltration, and paraffin embedding steps. Proper processing maintains morphology and antigen integrity for sectioning.

Lesson 4 • Quality Assessment of Processed Tissue

Evaluates hematoxylin and eosin morphology as a proxy for IHC readiness. Identifies processing artifacts that compromise staining quality.

Lesson 1 • Sectioning Troubleshooting

Identifies chatter, compression, and tearing artifacts and their causes. Systematic troubleshooting reduces wasted tissue and reagent costs.

Lesson 2 • Rotary Microtome Operation

Covers blade types, chuck alignment, and sectioning mechanics. Correct microtome setup is prerequisite to obtaining ribbon-quality sections.

Lesson 3 • Section Mounting and Adhesion

Teaches slide coating, water bath temperature, and drying protocols. Adequate adhesion prevents section loss during aqueous IHC steps.

Lesson 1 • Enzymatic Antigen Retrieval

Teaches protease-based retrieval using proteinase K, trypsin, and pepsin. Enzyme concentration and incubation time require careful titration per antibody.

Lesson 2 • Heat-Induced Epitope Retrieval

Covers buffer composition, pH, temperature, and heating devices for HIER. pH and temperature are the primary variables controlling retrieval efficiency.

Lesson 3 • Retrieval Optimization and Validation

Applies a systematic matrix approach to identify optimal retrieval conditions. Validated retrieval conditions are documented for reproducible routine use.

Lesson 1 • Antibody Validation Strategies

Applies genetic, orthogonal, and expression-based validation approaches. Validation evidence must demonstrate specificity before clinical or research use.

Lesson 2 • Antibody Storage and Quality Control

Addresses aliquoting, freeze-thaw cycles, and stability testing for antibodies. Improper storage degrades antibody performance and introduces batch variability.

Lesson 3 • Secondary Antibody and Detection Reagents

Covers secondary antibody species matching, conjugate types, and polymer systems. Mismatched secondaries produce false signals that invalidate results.

Lesson 4 • Primary Antibody Characteristics

Examines clone selection, host species, and target specificity for primary antibodies. Clone and species choices determine cross-reactivity and detection system compatibility.

Lesson 1 • Blocking Endogenous Interference

Teaches peroxidase quenching, biotin blocking, and serum blocking steps. Blocking eliminates non-specific background that obscures true signal.

Lesson 2 • Chromogen Application and Counterstaining

Applies DAB, AEC, and other chromogens with controlled development times. Counterstaining with hematoxylin provides morphological context for interpretation.

Lesson 3 • Deparaffinisation and Rehydration

Covers xylene substitutes, graded alcohols, and buffer equilibration steps. Complete deparaffinisation is essential for uniform reagent penetration.

Lesson 4 • Primary and Secondary Antibody Incubation

Optimises antibody dilution, incubation time, temperature, and humidity chamber use. Consistent incubation conditions are critical for reproducible staining intensity.

Lesson 1 • Corrective and Preventive Action

Applies CAPA methodology to IHC failures and documents resolutions. Preventive actions reduce recurrence and support continuous quality improvement.

Lesson 2 • Control Tissue Selection and Use

Defines positive, negative, and internal controls and their placement on each run. Appropriate controls are the primary safeguard against false results.

Lesson 3 • Recognising and Diagnosing Staining Failures

Catalogues no-signal, weak-signal, and high-background failure patterns with root causes. Systematic diagnosis prevents repeated failures and wasted reagents.

Lesson 4 • Proficiency Testing and External Quality Assessment

Participates in external quality assessment schemes to benchmark laboratory performance. EQA results drive targeted improvement in staining and interpretation.

Lesson 1 • Distinguishing True Signal from Artefact

Differentiates specific staining from edge, crush, and necrosis artefacts. Artefact recognition prevents misclassification of negative cases as positive.

Lesson 2 • IHC Report Writing and Communication

Structures IHC reports with clear result statements, interpretive comments, and caveats. Reports must convey actionable information to clinicians and multidisciplinary teams.

Lesson 3 • Principles of IHC Slide Evaluation

Establishes a structured approach to assessing staining pattern, intensity, and distribution. Systematic evaluation reduces inter-observer variability in routine practice.

Lesson 4 • Integrating IHC with Clinical Context

Correlates IHC findings with morphology, clinical history, and molecular data. Integration prevents over-reliance on IHC alone and improves diagnostic accuracy.