- Tissue Preparation
- Collect tissue samples and fix them in formalin to preserve protein structure and tissue morphology.
- Embed the samples in paraffin, cut them into thin sections (3-5 µm), and mount them on slides.
- Dewaxing and Antigen Retrieval
- Remove paraffin using xylene and rehydrate tissue sections with graded alcohol solutions.
- Perform antigen retrieval (e.g., heat-induced epitope retrieval) to restore accessibility to epitopes that were masked during fixation.
- Blocking
- Block endogenous peroxidase activity with hydrogen peroxide (if using enzyme-based detection).
- Use a blocking buffer (e.g., BSA or normal serum) to reduce nonspecific binding of antibodies.
- Primary Antibody Incubation
- Incubate tissue sections with a specific primary antibody against the protein of interest. Examples include:
- Nuclear proteins: Antibodies against transcription factors.
- Cytoplasmic proteins: Antibodies against signaling proteins or enzymes.
- Membrane proteins: Antibodies against receptors or adhesion molecules.
- Optimize antibody concentration and incubation time for precise localization.
- Incubate tissue sections with a specific primary antibody against the protein of interest. Examples include:
- Secondary Antibodies and Detection
- Apply secondary antibodies conjugated to enzymes (e.g., HRP) or fluorophores.
- Use chromogenic substrates (e.g., DAB) for light microscopy or fluorescence imaging.
- Counterstaining and Visualization
- Counterstain with hematoxylin to obtain structural context.
- Visualize sections under light or fluorescence microscopy.
- Applications
- Study cellular processes and protein functions.
- Identify mislocalized proteins associated with diseases.
- Explore dynamic protein changes during development and disease progression.
This structured approach ensures clear communication of the IHC process, making it easier to understand and follow.
