Immunohistochemistry (IHC) is a laboratory technique that uses the specific binding of an antibody to an antigen in a tissue section to visualize the location of a target protein. Unlike traditional histology, IHC provides cellular and subcellular localization of specific molecules. The frozen section protocol offers distinct advantages over formalin-fixed, paraffin-embedded (FFPE) tissues. Frozen sections are used when the protein of interest is sensitive to the chemical cross-linking and heat exposure involved in paraffin processing. This method bypasses harsh chemical steps, preserving the native structure of the protein, which often results in a stronger antibody-binding signal. The speed of the frozen section method also makes it invaluable for time-sensitive applications, such as rapid pathological diagnosis during surgery.
Preparing the Tissue for Sectioning
The initial handling of the tissue requires rapid cooling to prevent degradation and minimize ice crystal artifacts. Freshly harvested tissue blocks, typically no larger than $1.0 \times 1.0 \times 0.5$ cm, are immediately embedded in an inert water-soluble medium, such as Optimal Cutting Temperature (OCT) compound. This medium provides a supportive matrix during sectioning.
Optimal freezing is accomplished by “snap-freezing” the tissue to ensure the water content solidifies instantly, preventing the formation of large, destructive ice crystals. A common method involves submerging the OCT-embedded block into isopentane pre-chilled in liquid nitrogen to approximately $-80^\circ$C. Once frozen, the tissue blocks can be stored long-term at $-80^\circ$C until sectioning.
The frozen block is sectioned using a cryostat, a microtome housed within a freezer cabinet typically maintained between $-15^\circ$C and $-25^\circ$C. The exact temperature depends on the tissue type and its fat content, with fattier tissues requiring lower temperatures. Sections are typically cut to a thickness of 5 to 10 micrometers ($\mu$m).
After cutting, sections are immediately transferred to positively charged glass slides, which use electrostatic forces to help the tissue adhere. The slides are then allowed to air-dry for at least 30 minutes to promote robust adhesion. Cut slides should be stored in a sealed box at $-20^\circ$C or $-80^\circ$C to maintain antigen integrity until the staining protocol begins.
Essential Steps of the Staining Protocol
Once the frozen section is adhered to the slide, the first chemical step is fixation, performed after sectioning. Fixation is brief, typically five to ten minutes, using a cold organic solvent (e.g., acetone or ethanol) or a mild cross-linking agent like 4% paraformaldehyde (PFA). This stabilizes the cellular structure and immobilizes target proteins without masking antigen binding sites.
Following fixation, a gentle washing step with a buffered saline solution, such as phosphate-buffered saline (PBS), removes fixative residue. For intracellular targets, a permeabilization step is introduced using a mild detergent (e.g., 0.1% Triton X-100). This creates small pores in the cell membranes, allowing large antibody molecules to access the cell interior.
The next stage is blocking, which minimizes non-specific antibody binding and reduces background signal. The tissue is incubated with a blocking solution, often 5% to 10% normal serum derived from the secondary antibody’s host species. This serum saturates sticky sites on the tissue before the primary antibody is introduced.
The core of the protocol involves sequential incubation with primary and secondary antibodies. The primary antibody, which recognizes the protein of interest, is typically diluted in the blocking buffer and incubated with the tissue, often overnight at $4^\circ$C. After washing away unbound primary antibody, the secondary antibody is applied. This antibody binds to the primary antibody’s species and is conjugated to a detectable marker, such as a fluorophore or an enzyme.
Detection Methods and Final Preparation
Chromogenic Detection
Chromogenic detection uses a secondary antibody linked to an enzyme, most commonly horseradish peroxidase (HRP). HRP catalyzes a reaction with a substrate like 3,3′-Diaminobenzidine (DAB), producing a stable, insoluble brown precipitate at the site of the target protein. This signal is viewed using a standard brightfield microscope.
For chromogenic staining, endogenous peroxidase activity (e.g., in blood cells) must be quenched before primary antibody incubation to prevent false-positive staining. This is usually done by incubating the section in 3% hydrogen peroxide in methanol. Following color development, a counterstain, such as Hematoxylin, is applied to stain cell nuclei blue, providing morphological context.
Fluorescent Detection
Fluorescent detection, or immunofluorescence (IF), uses a secondary antibody conjugated to a fluorophore. A major advantage of IF is the ability to simultaneously visualize multiple proteins using different colored fluorophores, often using DAPI to counterstain the nucleus blue. Fluorescent signals are susceptible to photobleaching, the irreversible loss of fluorescence upon light exposure.
Mounting and Storage
The choice of mounting medium protects the stained section and optimizes imaging. Chromogenic slides require full dehydration and clearing before using a permanent, organic mounting medium (e.g., DPX) for long-term room-temperature storage. Fluorescently labeled sections must be mounted in an aqueous medium containing an anti-fade agent to retard photobleaching, and these slides must be stored at $4^\circ$C or $-20^\circ$C in the dark.
Identifying and Correcting Common Issues
A frequent problem is the physical detachment of the tissue section from the slide during wash and incubation steps. This often stems from insufficient air-drying after sectioning or overly aggressive washing. To prevent detachment, slides should air-dry completely for at least one hour before fixation, and positively charged slides must be used for enhanced tissue retention.
High background staining, where the entire section is stained, obscures the specific signal. This non-specific binding is caused by inadequate blocking or an antibody concentration that is too high. Resolving this requires increasing the concentration or incubation time of the blocking serum, or running a titration experiment to find the optimal, lower dilution of the primary antibody.
Poor tissue morphology, manifesting as structural distortions or holes, results from sub-optimal freezing. When tissue freezes too slowly, large ice crystals form within the cells, rupturing the cellular architecture. This is corrected by ensuring the tissue block is truly “snap-frozen,” using rapid cooling (e.g., isopentane chilled in liquid nitrogen) to promote the formation of tiny, non-damaging ice crystals.
If the chromogenic signal appears everywhere, it may be due to endogenous enzymes, such as peroxidase, that were not properly inhibited. For HRP-based detection, effective pre-treatment with hydrogen peroxide is mandatory to quench this background activity. If the specific signal is weak or absent, initial troubleshooting steps include confirming antibody compatibility and ensuring the primary antibody was not incubated too long at a cold temperature.