Immunohistochemistry (IHC) is a laboratory method using antibodies to detect and visualize specific molecules, typically proteins, within biological tissue samples. It relies on antibody-antigen binding, where an antibody attaches to its target, and a label (like a fluorescent dye or enzyme) creates a visible signal under a microscope.
Frozen sections are a tissue preparation approach for IHC, rapidly freezing tissue to preserve architecture and molecular integrity. This method offers speed and maintains cellular components altered by other techniques. IHC with frozen sections applies widely in scientific research and diagnostic pathology, aiding clinical decision-making and providing insights into disease mechanisms.
Preparing Tissue for Immunohistochemistry
Tissue preparation for frozen section IHC begins with rapid collection and freezing to prevent degradation and preserve cellular morphology. Freshly dissected tissue blocks, no more than 5 mm thick, are placed into embedding molds and filled with a cryoprotectant like Optimal Cutting Temperature (OCT) compound, which supports the tissue during freezing and sectioning.
To achieve rapid freezing, the OCT-immersed tissue block is submerged in liquid nitrogen or an isopentane bath cooled by liquid nitrogen. This quick freezing prevents large ice crystals that damage cellular structures. Frozen blocks can be stored at -80°C until sectioning.
Cryosectioning uses a cryostat, a refrigerated microtome (usually -15°C to -23°C). The frozen tissue block is mounted and cut into 5-15 µm thick sections. Maintaining low temperature ensures the tissue remains solid. Sections are transferred to specialized glass slides, often pre-coated with gelatin or poly-L-lysine for adhesion. Mounted sections are air-dried for 15-30 minutes to secure them before staining.
The Core Steps of Immunohistochemistry Staining
Once frozen tissue sections are prepared and mounted, the IHC staining protocol commences. The initial step is fixation, which stabilizes tissue and preserves target antigens for antibody binding. Common fixatives for frozen sections include cold acetone, methanol, ethanol, or 4% paraformaldehyde (PFA). Acetone fixation is performed at -20°C for 10-20 minutes; PFA fixation occurs at room temperature for 15 minutes.
Following fixation, a blocking step prevents non-specific antibody binding to tissue sites other than the target antigen, avoiding false-positive signals. This is achieved by incubating sections with a blocking solution (e.g., 1-10% normal serum from the secondary antibody’s species, or BSA). Incubation lasts 30-60 minutes at room temperature in a humidified chamber to prevent drying.
After blocking, the primary antibody is applied. This antibody specifically recognizes and binds to the target antigen. Diluted in an appropriate buffer (containing serum or BSA), it’s incubated with sections. Incubation times vary, but overnight at 4°C or 1-2 hours at room temperature is common, depending on the antibody and antigen.
After primary antibody incubation, slides are washed to remove unbound antibody. The secondary antibody is then applied. This antibody binds specifically to the primary antibody and is conjugated to a detection molecule (e.g., an enzyme like HRP, or a fluorophore). Secondary antibody incubation lasts 30 minutes to 1 hour at room temperature.
The detection step follows secondary antibody incubation, generating a visible signal from the conjugated label. If an enzyme like HRP is used, a chromogen substrate (e.g., 3,3′-diaminobenzidine (DAB)) reacts with the enzyme to produce a colored precipitate, typically brown, at the antigen’s location. For fluorophore-conjugated secondary antibodies, the signal is visualized directly with a fluorescence microscope.
Finally, counterstaining and mounting complete the protocol. Counterstaining, often with hematoxylin, provides a contrasting color to visualize tissue morphology and cellular nuclei, aiding antigen localization. After counterstaining, sections are dehydrated through alcohol washes, cleared with xylene, and mounted with a coverslip using an appropriate medium for permanent preservation and microscopic examination.
Interpreting Results and Applications
Interpreting IHC results on frozen sections involves microscopic examination to assess signal presence, location, and intensity. Stained slides show distinct color or fluorescence indicating target molecule presence. Staining intensity can also indicate target protein abundance. Pathologists and researchers analyze these patterns to understand cellular distribution and expression.
In diagnostic pathology, IHC on frozen sections is used for rapid diagnosis during surgical procedures. This allows surgeons to make immediate decisions, such as determining tumor margins for complete cancerous tissue removal or identifying tumor type. The speed of frozen section processing, yielding results within minutes, is an advantage in guiding real-time surgical interventions.
Beyond rapid diagnostics, IHC on frozen sections has numerous research applications. It helps understand disease mechanisms by localizing specific proteins in diseased tissues and aids drug discovery by identifying potential therapeutic targets. Researchers also use this technique in basic biological studies to map protein distribution within various cell types and tissues.
Frozen sections are preferred over formalin-fixed paraffin-embedded (FFPE) tissues for certain applications because they better preserve enzyme activity, lipids, and antigens sensitive to chemical processing in paraffin embedding. This preservation makes frozen sections valuable for studying delicate molecules and for rapid intraoperative analysis, offering a direct and timely view into tissue pathology and molecular expression.