Tissue and cell samples must be appropriately harvested and prepared for each IHC/ICC study. To facilitate the required incubation steps, whole tissues must be cut into ultra thin (5-10 µm) sections or cut into smaller pieces for whole mount IHC. For ICC experiments, cells must be attached to a microscope slide or coverslip before commencing the staining procedure. Sample preparation is also intimately linked to the method of fixation, which in turn is influenced by the desired detection technique (fluorescence versus chromogenic). In most circumstances, one experimental variable will determine the most appropriate method of sample preparation. For example, tissue that is immersion-fixed in formaldehyde must be paraffin-embedded and cut using a microtome. Alternatively, to detect a phosphorylation-dependent epitope, tissue may need to be snap frozen, which requires a cryostat for sample sectioning followed by alcohol fixation.
Paraffin embedding offers the best option for long-term preservation of tissue samples. Tissue must be fixed before being embedded in paraffin. Fixation can be achieved by perfusion or immersion immediately following dissection, and typically requires 4-24 hours. It is not recommended to fix tissue for more than 24 hours because overfixation can cause masking of the antigen. If necessary, tissues can be transferred to alcohol after fixation until there is time for the embedding process.
Because paraffin is immiscible with water, tissue must be dehydrated before adding molten paraffin wax. Dehydration is achieved by immersion in increasing concentrations of alcohol. This approach allows for a gradual change in hydrophobicity and minimizes cell damage. Following dehydration, the tissue is incubated with xylene to clear any remaining ethanol. Paraffin is typically heated to 60 °C for embedding and is subsequently allowed to harden overnight. The tissue is subsequently cut with a sharp blade into ultra thin slices using a microtome. Sections are then dried onto microscope slides and can be stored at room temperature for extended periods of time. Tissue sections must be rehydrated before commencing the IHC/ICC protocol. Paraffin is the cheapest and most commonly used substance for tissue embedding. However, tissue can also be embedded in plastic, which sets harder and allows thinner tissue sections (1.5 mm versus 5 mm).
One of the benefits of frozen tissue samples is the time saved by omitting the initial fixation step required for paraffin-embedded tissue. Snap freezing can be particularly beneficial when detecting post-translational modifications such as phosphorylation. Frozen tissue can be prepared by immersing the tissue in liquid nitrogen or isopentane, or by burying it in dry ice. For frozen samples, a short fixation is done after freezing and sectioning. Frozen tissue sections are most often fixed using alcohol, which avoids the possible requirement to retrieve epitopes masked by formaldehyde cross-linking. Frozen tissue is cut on a cryostat and sections can be stored at -80 °C for up to 1 year.
Frozen tissue sections can be processed in a shorter amount of time than paraffin-embedded sections. However, freezing is not adequate for long-term preservation of the tissues and the formation of ice crystals within the cells may negatively affect subcellular detail. In addition, frozen sections are often thicker than paraffin sections. This can increase the potential for lower microscopic resolution and poor images of tissue morphology. Because frozen tissue often retains enzymatic activities, blocking the functions of endogenous enzymes that may affect the IHC detection method is also particularly important.
IHC Sample Preparation Comparison
||Multiple years at room temperature
||1 year at -80 °C
||Preserves tissue morphology
||Preserves enzyme & antigen function
||Overfixation can mask the epitope
||Formation of ice crystals may negatively affect tissue structure
To perform ICC studies, the cells must first be attached to a solid support, such as a microscope slide or coverslip. This is simple with adherent cells, which can be grown directly on high-grade, sterilized coverslips placed on the bottom of a 6- or 24-well plate. Cell adhesion can be enhanced by pre-coating the coverslip with a charged polymer like Poly-L-Lysine and/or an extracellular matrix protein such as Laminin, Fibronectin, or Collagen. All steps of the staining process can be performed with the coverslip in the microplate by adding the appropriate solution to each well and removing solutions by aspiration. Cells in suspension can be adhered to a slide by Cytospin® centrifugation or by a 10 minute incubation of the cells on slides that have been coated with Poly-L-Lysine.
Note: It is important to be able to recognize artifacts of poorly prepared samples in order to properly interpret results. For example, folds or tears in the tissue, or bubbles between the tissue/cells and the slide, can collect staining reagents non-specifically and obscure positive staining or lead to false-positive signals.
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