IHC/ICC/IF, silly points are not clear? They are all immunodetection techniques for localization of antigen expression. Because of their relatively simple and intuitive technology, they have been widely used in scientific research and clinical practice. However, there are many variables that affect the final detection effect ; each specific test encounters multiple variables that need to be adjusted. Specifically, which method should be used to preserve the tissue sample; whether the fixed specimen should be alcohol or aldehyde; how to choose the most suitable primary antibody; the specific methods and conditions for antigen retrieval should be considered.
Therefore, before starting the experiment, it is best to master the common sense of the following groups of immunoassay techniques. The ability to distinguish their characteristics will enable you to make better experimental designs and improve the success rate of the experiment.
Paraffin section VS frozen section
â–¶ Paraffin section
Paraffin embedding is the cheapest and commonly used method of tissue embedding for specimens that require long-term preservation. The sample must be fixed before embedding, and the fixation time is usually 4 to 24 hours. It is necessary to consider that the antigen caused by excessive fixation is blocked. In the case where it cannot be immediately buried, the tissue after the fixation is completed can be temporarily stored in the ethanol. If a resin is used instead of paraffin as an embedding agent, a thinner tissue section (1.5 μm vs. 5 μm) can be obtained. There are many steps in paraffin sectioning, and the antigen activity in the tablet is reduced, but the tissue structure and cell morphology are clear, which is a conventional method for preparing immunohistochemistry.
â–¶ Frozen slices
Frozen sections cannot be preserved for a long time, and the formation of ice crystals during freezing tends to affect the subcellular structure, making the tissue structure less clear than paraffin sections. Frozen tissue sections can be stored at -80 ° C for one year. The frozen sectioning step is simple and rapid, and the sample can be directly frozen with liquid nitrogen or liquid isopentane, dry ice, etc. without fixing. After freezing and sectioning, the sections were fixed with ethanol to avoid cross-linking of the epitope by formaldehyde without antigen retrieval.
Frozen sections are a good choice when testing cell membrane surface antigens and hydrolases such as phosphorylation or some of the poor tolerance to organic solvents or heat.
Formaldehyde fixed VS alcohol fixation
â–¶ Formaldehyde fixation
Formaldehyde is the most commonly used fixative for protein preservation. Formaldehyde can form crosslinks between a variety of protein end groups to achieve a fixed effect. However, formaldehyde fixation alters the epitope and prevents antibody binding. Therefore, enzymatic digestion or thermal antigen retrieval is often used to break the aldehyde bond of the protein cross-linked with formaldehyde for subsequent staining. In the detection of phosphate-dependent epitopes, it is more appropriate to use ice-cold anhydrous methanol or absolute ethanol for immobilization.
â–¶ Alcohol fixation
Commonly used alcohol fixatives are methanol and ethanol. The principle of immobilization is to compete with water for hydrogen bonds in proteins, thereby replacing water molecules in tissues and stabilizing the secondary structure while affecting the tertiary structure of proteins. Alcohols dissolve lipids and shrink to tissues, so it is generally believed that alcohol fixation is not conducive to protecting tissue shape. Because alcohol fixation will form a layer of protein on the surface of the tissue, this layer will affect the penetration of the fixative, resulting in poor tissue fixation. Therefore, alcohol is usually used to fix frozen tissue sections and cells, which is more suitable for Cell membrane surface antigen. Antigen retrieval is not recommended after alcohol fixation to avoid damage to sample integrity.
Tissue fixation VS cell fixation
â–¶ Tissue fixation
Whole animal perfusion fixation is often the best method for preserving antigens and is suitable for the study of intact tissues in mice, rats and guinea pigs. The exfoliated tissue can be fixed by soaking with a fixative. A commonly used fixative is a 4% formaldehyde solution in PBS. Tissues with a thickness of 10 mm are more favorable for penetration of the fixative. To enhance the penetration of the fixative. For complete fixation, the volume of the fixative is at least 50 to 100 times the volume of the tissue.
â–¶ Cell fixation
Cell fixation is shorter than tissue fixation and a lower concentration of fixative can be used. In general, fixing with 2% formaldehyde solution for 20 minutes at room temperature is sufficient to preserve cell morphology and antigenicity. Most of the cells can be removed by removing the medium and then adding a fixative. For some cells that are damaged by surface tension after removing the medium, the cells can be pre-fixed by directly adding an equal volume of fixative to the medium. After two minutes, remove the pre-fixed medium and add fresh 2% fixative to complete the fixation.
Heat-induced repair VS enzyme induced repair
â–¶ Heat-induced epitope repair (HIER)
Factors such as antigen, antibody, tissue type, fixation method, and duration need to be considered in the antigen retrieval process, so it is necessary to optimize the repair program for each factor. Commonly used heat-induced epitope repairs can be carried out with a variety of heating equipment. Microwave ovens are the most convenient equipment. Choosing the right heat repair buffer can achieve good repair results at the right temperature and time. These conditions are based on experience. To determine. In the HIER method, the most important point is that the slides are naturally cooled after the repair is completed, because forced cooling makes it difficult to restore the original configuration of the protein.
â–¶ Protease-induced epitope repair (PIER)
The mechanism of protease-induced epitope repair is that the enzyme cleaves peptides that may mask the epitope, and is usually repaired by proteinase K, trypsin and pepsin. The PIER method may destroy tissue morphology and target antigen, and the success rate is usually low. Less used.
Polyclonal antibodies recognize multiple epitopes compared to monoclonal antibodies, and the possibility of detecting antigens is high even without antigen retrieval.
Monoclonal primary antibody VS polyclonal primary antibody
Monoclonal antibodies have high affinity and high specificity for a single antigenic epitope due to their originating from a single B cell. Monoclonal antibodies bind best when the antigen retains its native conformation, and once the antigenic conformation changes, the binding effect is greatly compromised. Therefore, the binding of the monoclonal antibody to the antigen is easily interfered by factors such as temperature, pH, fixation, salt concentration, post-translational modification, and protein interaction.
Polyclonal antibodies recognize multiple epitopes, so they are less affected by protein conformational changes. Generally, polyclonal antibodies are also more stable under the influence of pH changes and salt concentrations than monoclonal antibodies. Therefore, more polyclonal antibodies were selected in the IHC/ICC experiment.
Direct detection VS indirect detection
Highly expressed antigens can generally be achieved by direct detection. Direct detection is not interfered with by non-specific binding of the secondary antibody, and the operation is simpler when performing multicolor detection. However, the sensitivity of the assay is limited and there is no power for the low expressed antigen, so the application of this method is limited by the abundance of the antigen.
The indirect detection method amplifies the detection signal by combining each primary antibody with at least two secondary antibodies, so that the detection limit is lower and a stronger signal can be generated. However, closure and control were added due to the need to bind secondary antibodies.
The most common biotin-Avidin-System (BAS) is available in combination with a variety of markers currently available for research. The strong combination of high affinity between biotin and avidin and multi-stage amplification effect make BAS immunolabeling and related tracer analysis more sensitive. Currently widely used in micro-antigen, antibody qualitative, quantitative detection and localization observation research. Streptavidin, like avidin, binds 4 molecules of biotin per molecule, while streptavidin does not contain any glycosyl groups, avoiding the non-specific binding of the avidin to the cell surface polysaccharides. Therefore, incubation with avidin or streptavidin with a biotinylated secondary antibody can significantly amplify the signal.
Fluorescence detection VS color detection
â–¶ Fluorescence detection
Fluorescence detection is based on the use of fluorochromes that illuminate when excited by light of shorter wavelengths. The fluorochrome can be directly bound to the primary or secondary antibody or to streptavidin. Immunofluorescence is often used for simultaneous observation of multiple cellular targets. For example, the tissue can be incubated with a mixture of different primary antibodies and then incubated with a secondary antibody that incorporates a fluorochrome that illuminates at different wavelengths.
Multicolor experiments must be designed to limit the cross-reactivity between the detection reagents and the spectral properties of the fluorescent dyes used. In order to limit cross-reactivity between secondary antibodies, primary antibodies should be from different species. This allows the use of species-specific secondary antibodies, each identifying only one primary antibody.
For convenience of color detection, primary, secondary or streptavidin can be combined with an enzyme. Upon addition of a soluble organic substrate, the enzyme reacts with the substrate to produce an insoluble colored product that deposits at the site of antigen expression. Commonly used enzymes include horseradish peroxidase (HRP) and alkaline phosphatase (AP), which combine 3,3' diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC), respectively. Converted to the final product of brown and red. DAB is more commonly used than AEC because it is soluble in alcohol and is more susceptible to fading when exposed to excessive light. Water-based counterstains and sealers must be used when using AEC.
Color detection is generally considered more sensitive than immunofluorescence, but it is less convenient because of the more incubation and blocking steps. Similar to immunofluorescence, chromogenic detection also allows observation of multiple antigens, but these antigens can only be in different locations in cells and tissues, as overlapping colors may make the results difficult to interpret.
Source R&DSystems
Related Links:
- Immunohistochemistry (IHC)
- Immunofluorescence (IF)
- Laser confocal
- Cell experiments (flow, proliferation, invasion, electron microscopy)
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