2 Corresponding author: William H. Yong M.D., Brain Tumor Translational Resource, David Geffen School of Medicine at UCLA, CHS13-145B, 10833 Le Conte Avenue, Los Angeles, CA, USA, Phone: (310) 825-8269, ude.alcu.tendem@gnoYW
The publisher's final edited version of this article is available at Methods Mol BiolImmunofluorescence (IF) is an important immunochemical technique that allows detection and localization of a wide variety of antigens in different types of tissues of various cell preparations. IF allows for excellent sensitivity and amplification of signal in comparison to immunohistochemistry, employing various microscopy techniques. There are two methods available, depending on the scope of the experiment or the specific antibodies in use: Direct (Primary) or Indirect (Secondary). Here, we describe preparation of specimens preserved in different types of media and step-by-step methods for both direct and indirect immunofluorescence staining.
Keywords: Immunofluorescence, Immunohistochemistry, Immunocytochemistry, Fixation, Antigen Retrieval, Fluorescence, Fluorophore
Immunofluorescence (IF) is a technique that permits visualization of virtually many components in any given tissue or cell type. This broad capability is achieved through combinations of specific antibodies tagged with fluorophores. Consequently, the possible applications in research and patient care are numerous. For IF evaluation, a variety of sample conditions can be employed. It can be performed on cultured cells or cell suspensions, as well as on specific targets in tissues samples or entire organisms. Fresh samples can also be used in IF studies if they are snap frozen or placed in Michel’s transport medium, which allows transportation and storage at room temperature for up to 72 hours.
Fixation is an essential preliminary step in IF staining in order to prevent autolysis, mitigate putrefaction, and preserve morphology while maintaining antigenicity. The ideal fixation method serves to immobilize target antigens without disturbing cellular architecture to allow antibodies maximum access to any targeted cellular components. It is possible that a given fixative may adequately preserve the immunoreactivity of a particular epitope, while degrading or masking other epitopes within the same protein. Consequently, as there is no universal fixative for every antigen, optimal fixatives and fixation methods may need to be determined empirically based on the given antigen and sample type. Chemical fixatives include cross-linking reagents and organic solvents, used separately or in combination. Cross-linking (additive) reagents bind to cellular and tissue components by addition, forming intra and intermolecular methylene cross-links. Common examples include formaldehyde and glutaraldehyde. Organic solvents work to remove lipids and dehydrate cells, denaturing and precipitating cellular components. In addition, by permeabilizing cell membranes, organic solvents eliminate the need for detergent treatments. Common examples include methanol and acetone (1).
Following fixation, tissues are often embedded into paraffin in order to solidify the sample for sectioning. Thin slices embedded in a hard matrix allow for dyes, probes, and antibodies to effective reach target sites without the obstruction of multiple cell layers. Paraffin blocks can be stored at room temperature, but they must be kept in the dark under moisture-controlled conditions. Embedded tissue blocks should be considered infectious material, ensuring all proper personal protective equipment is worn when handling or working with blocks. Once paraffin sections have been cut and mounted onto glass slides, however, they must be deparaffinated and rehydrated so that only the tissue section remains on the slide for subsequent antigen retrieval and IF procedures. Xylene is the most common medium for deparaffinization, followed with washes of ethanol and distilled water for rehydration (2).
Antigen retrieval is necessary to restore epitope-antibody reactivity. Reactivity is altered during fixation, in which proteins undergo structural modification, forming cross-links that can mask the target epitope. This is especially prevalent with the use of additive reagents. The need for antigen retrieval is dictated by various factors: target antigen identity, antibody character, tissue type, the method and duration of fixation. Antigen demonstration levels can be recovered using retrieval agents to cleave the cross-links formed during fixation. There are two main methods of antigen retrieval: Protease-Induced Epitope Retrieval (PIER) and Heat-Induced Epitope Retrieval (HIER). As with any technique, both methods must be optimized prior to any application.
In PIER, enzymes serve as the primary method in restoring antigenicity. Though the exact mechanism is not yet fully understood, it is generally thought that the enzymes cleave the protein cross-links to unmask the target epitopes. Common examples of proteases include Proteinase K, Trypsin, Pepsin, and Pronase. The specific enzyme to be used should be detailed in any antibody data sheet provided by the manufacturer. However, there are several disadvantages to the PIER method. Because the enzymes used for PIER cannot target only the exact protein cross-links present in the fixed tissue sample, non-specific enzyme digestion can potentially destroy tissue morphology and any antigens of interest (3). Consequently, strict incubation times and optimal enzyme concentrations must be determined beforehand. Much higher rates of restoring immunoreactivity can be achieved with the HIER method.
With HIER, heat and pressure are used to restore antigenicity. Just as in the case of PIER, the exact mechanism of reversing protein cross-links is unknown, though it is assumed secondary and tertiary protein structure must be restored for the epitope to be unmasked. In general, the HIER method involves heating the mounted tissue sample in a buffer solution, with heat to cleave cross-links and buffer to maintain protein conformation. Buffer solutions can be categorized into low, neutral, and high pH solution compositions. Low pH solutions are usually buffered with glycine-HCl, neutral pH solutions with citric acid, and high pH solutions with Tris or EDTA. Though high pH solutions usually serve as the most effective buffers, high pH and EDTA have higher chances of convoluting tissue and cellular morphology (4). As different HIER methods vary upon combinations of appliance type, temperature, duration, and pH, the optimal method and specifications must be determined by conducting preliminary control studies. In addition, an HIER device should be chosen based on the following factors: temperature range, buffer volume, minimal evaporation, and minimal boil-over. A variety of commercial HIER devices are available to meet these requirements, though there are no set industry standards or specialized manufacturers (5).
Following fixation and antigen retrieval, either of two IF methods can be employed: Direct (Primary) IF or Indirect (Secondary) IF. In the direct method, fluorophore label is conjugated directly to the primary antibody that will be reacting with the target epitope (see Figure 1 ). The indirect method involves a two-step incubation process: 1) a primary antibody binds to the target epitope, 2) a fluorophore-tagged secondary antibody recognizes and binds to the primary antibody (see Figure 2 ). Although the direct method is quicker, the indirect method is more widely employed for its high sensitivity, signal amplification, and its ability to detect several targets in the same sample (6, 7). In choosing the proper antibodies, several criteria must be taken into consideration. To prevent the secondary antibody from cross-reacting with endogenous immunoglobulins in the tissue sample, the primary antibody should be derived from a different species than that of the sample. Consequently, the secondary antibody must be against the host species of the primary antibody (8). Secondary antibodies can be modified and conjugated in multiple ways for purposes of visualization and signal amplification. Commonly, secondary antibodies are conjugated with fluorescent labels which emit upon photoexcitation. Enzymatic labels can also be conjugated to react with chromogen substrates for colorimetric analyses. For greater signal amplification, polyclonal or biotinylated secondary antibodies can be employed. For each primary antibody, polyclonal secondary antibodies are able to recognize multiple epitopes to increase binding and signal levels (see Figure 3 ) (9). Similarly, multiple fluorochrome-protein (avidin or streptavidin) complexes can bind to a single biotinylated secondary antibody to increase signal levels (see Figure 4 ). A combination of these methods can be used as well for greater signal amplification, as detailed later on.