This can be effective if the antigenic site is blocked by another protein and removing it increases access. The avidin-biotin-peroxidase method suffers from high background staining due to binding of endogenous biotin, and the method is now largely obsolete. Immerse in 2 changes of deionized water and let sit in deionized water for 5 minutes. Detection was performed using an HRP-conjugated secondary antibody followed by chromogenic detection using DAB as the substrate. A high primary antibody concentration will increase these interactions and thus increase nonspecific binding and background staining. Your detection methods can be either chromogenic, using secondary antibodies that are enzyme-labeled (eg, HRP, AP), or fluorescent (immunofluorescence) using secondary antibodies that are fluorochrome-labeled (eg, FITC, R-PE, Alexa-Fluor). At first glance it appears a straightforward procedure. . This technique, first described by Shi et al, has significantly increased the sensitivity of IHC and consequently greatly expanded its application (3, 13). et al. The use of an antibody that is known not to bind to the sample can be used, however this can be problematic if it binds non-specifically and yields a false positive. Solution:Ensure that the antibody diluent pH is within the specified range for optimum antibody binding (7.0 to 8.2) and that the antibody is stored according to the manufacturer's instructions. Guidelines for the standardization and analytic validation of immunohistochemical tests have been established by the College of American Pathologists (1, 11). 10X Antigen Decloaker (Biocare, Pacheco, CA, USA) diluted 1:10 with deionized water, 3% hydrogen peroxide (for blocking endogenous peroxidase); can also use a ready-to-use peroxidase blocking solution available from various manufacturers, Blocking reagent to decrease nonspecific background staining e.g. Sample fixation is key to tissue processing and is critical to prevent the degradation of antigens, cells, and tissue. Shi was able to unmask antigenic sites by heating deparaffinized tissue sections in a microwave oven and immersing them in metal salt solutions until the solutions reached the boiling point. Finally the pH of the substrate buffer must be appropriate for that specific substrate. IHC can be used in both transmitted light and fluorescence microscopy, and it can be used in combinations to identify and mark multiple proteins of interest. Background Sniper (Biocare, Pacheco, CA, USA), Charged or adhesion slides to promote tissue retention onto the slide. IHC image of Iron Stain Kit (ab150674) in formalin-fixed-paraffin embedded human liver. Let sit in deionized water for 5 min (slides can remain in deionized water for up to 30 minutes). brain tissue is usually cut thicker compared to other types of tissue) and mount on adhesion treated slides. Protein expression patterns are tremendously valuable for pathologists and as diagnostic tools. Sadly, the one that is most frequently cited in the literature may also be one of the most problematic. This can be done in any culture chamber with bottoms that are suitable for imaging, or using cells grown on cover slips in plastic multi-well plates that can subsequently be inverted and mounted on slides when the staining is finished. If there isautofluorescencein the test sample, then this suggests that either the tissue sample shows inherentautofluorescence(which is common) or that the fixation method is causingthe sample toautofluoresce. If your antibody is an HRP primary conjugate, then this block needs to be done before the addition of the primary antibody. Immunofluorescence techniques using fluorescent compounds are also available but require a fluorescence microscope (2). They may also be used as blocking reagents for Western blotting, IHC and IF.ChromPure proteins are derived from the serum of non-immunized animals and do not recognize any known antigens. The frightening use of 10% crude serum, found in many current protocols, is not only overkill but actively detrimental. Add CaCl 2 to the trypsin stock solution for a final concentration of 0.001%. Prepare antigen retrieval buffer (10X Biocare Antigen Decloaker diluted 1:10 with deionized water). pH 6 buffers are often used, but high pH buffers are widely applicable. National Library of Medicine The method is most commonly performed on formalin fixed paraffin embedded (FFPE) tissue which has the advantage of being amenable to easy storage, although it was first developed on frozen sections and can also be done on plastic embedded tissue (7, 8). If paraffin sections are used, deparaffinization and re-hydration must be done to prepare the sections for IHC. Sections are then allowed to cool for 20minutes and then heated a second time before cooling again, rinsing, and proceeding to IHC. You may need to optimize the concentration and incubation time. IHC secondary antibody and staining reagents are ancillary reagents to primary IHC antibodies. Some precipitates are photostable (HRP/DAB is very photostable, but HRP/AEC fades in sunlight), potentially allowing storage of the slides for many years. Secondary antibodies: labeled such as with an enzyme (e.g. 1Department of Pathology and Laboratory Medicine (Neuropathology), David Geffen School of Medicine at UCLA, Los Angeles, CA, USA, 2Brain Research Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA, 3Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA. Immunohistochemistry - Wikipedia The primary antibody, which can be monoclonal or polyclonal, is titrated to optimize contrast between positively staining tissue and nonspecific background staining, with the highest primary antibody dilution to prevent waste (1, 7). The secondary antibody should be phylogenetically as far apart as possible from a species from which your sample is derived. Frozen sections and floating sections are other options each method has advantages and limitations (Table 1). Autofluorescence levels and strange artifacts have both been observed when crude serum was used for blocking. A standard tool in many fields in the research setting, IHC has become an essential ancillary technique in clinical diagnostics in anatomic pathology (1) with the advent of antigen retrieval methods allowing it to be performed conveniently on formalin fixed paraffin embedded (FFPE) tissue (2, 3) and automated methods for high volume processing with reproducibility (4). The most conclusive demonstration of antibody specificity is lack of staining in tissues or cells in which the target protein has been knocked out. This prevents contamination or loss of the whole vial of antibody if a problem arises. Dako Pap Pen) draw a barrier around the tissue leaving some space between the tissue and barrier. Polyclonal antibodies provide a stronger chance of recognizing at least some epitopes in a sample, albeit with a higher possibility of recognizing an epitope that is not unique to a specific protein, thus generating a false positive result. Chromogenic Detection for Western Blot, IHC, and ELISA - Jackson Immuno Less useful than a monoclonal antibody for probing specific domains on an antigen. Not for use in diagnostic procedures. Although less quantitative than assays such as western blotting or ELISA, IHC gives invaluable information about protein localization in the context of intact tissue. Purchase these through your usual distributor. Heat mediated antigen retrieval was performed using Tris/EDTA buffer, pH 9.0 (ab93684). Lack of staining in tissues or cells known not to express the protein. Immunohistochemistry (IHC) Protocol | Rockland For specifics and timings please refer to the text. I will address each step of a typical fluorescence IHC protocol and provide the rationale for the step as well as suggestions for optimal results. This was all thrown into dispute when, in 1991, Shan-Rong Shi published a method to retrieve antigenic sites [3]. If incorrect, please enter your country/region into the box below, to view site information related to your country/region. Validated in WB, IP, IHC, ICC/IF, Flow Cyt (Intra) and tested in Mouse. Test the primary antibody for potency by staining tissue samples known to contain the target antigen with various concentrations of the primary antibody; do the test concurrently with the test sample. Customized products and commercial partnerships to accelerate your diagnostic and therapeutic programs. If a fluorescent marker is being used, check to make sure that there is noautofluorescencein the unprocessed, fixed tissue. Immunohistochemistry for Pathologists: Protocols, Pitfalls, and Tips It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide, This PDF is available to Subscribers Only. During an in-depth interview with the scientists at Acepix, IHC expert Dr. Nan Su revealed secretes on secondary antibody incubation time ihc, monoclonal vs polyclonal antibodies ihc, ihc antibody dilution, ihc antibody concentration, negative control ihc. The antibody may not be suitable for IHC procedures which reveal the protein in its native (3D form). An additional choice that must be made when selecting an antibody is whether to use a polyclonal or monoclonal antibody. Discover your comprehensive guide to immunohistochemistry (IHC). Use 10X PBS, pH 7.2 (0.2 M Potassium Phosphate, 1.5 M NaCl). A separate nuclear staining step is very fast (515minute incubation followed by three rinses of one minute each), and the results will be much more controllable. Background staining may be due to nonspecific antibody binding, more common in polyclonal antibodies, and endogenous peroxidase activity, more problematic in tissues with abundant hematopoietic elements such as bone marrow. The preparation of a well-stained sample is essential to the final result, because even the most high-tech microscope cannot make a quality image from a bad sample. The https:// ensures that you are connecting to the Immerse slides in 3 washes of xylene, each for 10 min for a total of 30 minutes (change solutions after they have been used for approximately 30 slides). FOIA To prevent contamination of your antibody solutions, wear gloves when dispensing antibodies, and use sterile pipette tips, if appropriate. While monoclonals do provide more specificity if the epitope is altered during fixation and sample processing, or is buried deep in the proteins tertiary structure, a monoclonal antibody may no longer recognize the epitope and result in a false negative result. The previous paragraph assumes the use of a tagged anti-IgG secondary antibody, but other variations are possible. The immune reactive products can be visualized by a marker including fluorescent dye, enzyme in general; radioactive element or colloidal gold also can be used. Customized products and commercial partnerships to accelerate your diagnostic and therapeutic programs. Anti-Tau antibody [EPR26001-28 . If the tissues turn brown, endogenous peroxidase (found in red blood cells, for example, which are generally within vessels within the tissue) is present and you require a blocking step. To avoid this happening to an introduced antibody, samples are usually incubated with a non-fluorescent, non-specific protein. Solution:Reduce the concentration of the secondary antibody. Federal government websites often end in .gov or .mil. Green is anti-NeuN [EPR12763] (ab177487), with goat anti-rabbit IgG conjugated to Alexa Fluor 488 (ab150097), red is anti-GFAP (ab4674), with goat anti-chicken IgY conjugated to Alexa Fluor 594 (ab150176). It can also be employed in multi-well plates on free-floating sections. In part 1 of this series [1], the fixation and sectioning of tissues for IHC was discussed. and transmitted securely. Generalized diagram of an IgG antibody showing the whole IgG, F(ab)2 and Fab fragments that result from pepsin and papain digestion. Avidin/biotin amplification. Updated May 10, 2022. The site is secure. In IHC, positive and negative controls are essential in order to correctly interpret the results [2]. Likely to cross-react and generate non-specific signal*. Again, for the brain slice rinsing 3 20minutes will be effective even after a prolonged primary incubation. . Commercial mounting media come in both liquid and self-hardening formulations. Tissue can be tested for endogenous AP by incubating with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium chloride (BCIP/NBT); if a blue color appears, then endogenous AP is present, and blocking is necessary. Using a biotinylated secondary antibody followed by a tagged avidin conjugate will cause signal amplification through the building up of numerous avidin/biotin complexes (Figure 3). IHC staining of paraffin-embedded wild type (A) and GSDMD KO mouse small intestine (B) with anti-GSDMD antibody [EPR20859] (ab219800) and HRP-polymer conjugated secondary antibody used in our micro-polymer IHC detection kits. However, it is hard to distinguish more than two chromogens on a slide, particularly if any chromogens overlay each other. More tolerant of minor changes in the antigen (eg polymorphism, heterogeneity of glycosylation, or slight denaturation). So, incubating for, say, two hours in a low concentration of secondary antibody will end up taking almost as . As it is not usually possible to change the antigen concentration, the optimal working concentration of each antibody must be determined with dilutions for each application and set of experimental conditions. Secondary Antibody; Unsurprisingly, this is the second antibody you apply, which binds to the first antibody and is conjugated to a fluorophore. See also the additional notes sections at the bottom of this page for more information. Traditional chromogenic mIHC relies on each antibody being raised in a different species or of a different isotype. Ensure gasket properly positioned on the removable pan. However, in practice, an IHC protocol has a number of steps where problems can occur, resulting in either false positive or false negative data. IHC experiments are always a balance between several factors that will produce a clearly labeled sample with low non-specific background staining. official website and that any information you provide is encrypted If these approaches are not sufficient to reduceautofluorescencewhile maintaining tissue sample detection, then the only other alternative is to just choose a fluorescent marker that will not compete with theautofluorescence. Gentler epitope retrieval and more definable parameters. Useful for epitopes that are difficult to retrieve. For example, if the recommended dilution is 1:400, then testing serial dilutions of 1:200, 1:400, and 1:800 is likely to show optimal signal to background staining on one of the dilutions. Here we describe how to define the optimal antibody concentration by titration and provide suggested dilutions for antibodies without recommended dilution on the datasheet. . Generalized flow chart of steps involved in immunohistochemical labeling. In immunohistochemical techniques, there are several steps prior to the final staining of the tissue antigen, and many potential problems can affect the outcome of the procedure. It preserves the antigen binding site on the primary antibody by conjugating to the secondary antibody. Good positive controls include a western blot showing that the antibody reacts appropriately and specifically with the purified protein of interest. Dust and debris are frequently a source of non-specific fluorescent background, and using clean, good-quality slides and cover slips are trivial investments in time and money that will pay off in improved results. A too high concentration or too long incubation time of the antibody or chromogen may also result in false positive or excessive background staining. The most common methods to increase the number of markers further use: a) spectral unmixing microscopes that enable more fluorescent dyes to be distinguished; and b) sequential antibody stripping and staining methods, often with tyramide signal amplification. A serum matching the species of the secondary antibody is an excellent blocking reagent. Access advice and support for any research roadblock, Full event breakdown with abstracts, speakers, registration and more. Multi-color fluorescent IHC staining of neonatal pancreas in mice using collagen IV (yellow), insulin (green), and glucagon (red) primary antibodies, and Cy2, Cy5 and Texas Red-conjugated secondary antibodies. Diagram of indirect antibody labeling where the labeled secondary antibody is bound to the primary antibody. Immerse slides in 3% hydrogen peroxide solution in Coplin jar for 5 minutes (can also use a ready-to-use peroxidase blocking solution). More challenging to image smaller structures and individual cells. The final step in any IHC protocol is to rinse the tissue section thoroughly, again 3 20minutes is advised, and if un-mounted, to place it between a clean glass slide and cover slip or into a viewing chamber or multi-well plate for microscopic observation. Solutions of 10% buffered formalin and 4% formaldehyde (also called paraformaldehyde) are typical fixatives these are near identical; formalin is a 40% solution of formaldehyde. Antibody Dilution Buffer. Unpurified antibody preparations differ significantly in antibody concentration. Add 500mL deionized water to pressure cooker and submerge heat shield, positioning in center of pan. However, this F(ab) approach does not produce a complete block and does leave some background. The use of high concentrations of antibody will also significantly increase the cost of the experiment. IHC ofhuman kidney tissue labeled with rabbit monoclonal anti-ZO1 (ab221547). Study of TNG260 and an Anti-PD Antibody in STK11 Mutated Solid Tumors It can be used in all subsequent steps including as a diluent for the antibodies. Specializing in Secondary Antibodies and Conjugates - For Western Blotting, IHC, ICC, Flow Cytometry, ELISA and other immunological applications. Table 3. One advantage of chromogens is that you can use them with an organic mounting medium, which tends to have a better refractive index, resulting in sharper images. Others have successfully employed antigen retrieval by using high or low pH buffers in place of the metal salts. When using horseradish peroxidase (HRP)-conjugated antibodies for detection, non-specific or high background staining may occur due to endogenous peroxidase activity. Apply species specific secondary antibody onto slides according to manufacturer specifications [e.g. A strong background signal suggests interference from endogenous peroxidases or phosphatases. All rights reserved. Immunohistochemistry (IHC) is the use of antibodies as probes to determine the localization of proteins in a cell or tissue sample. ChromPure proteins are primarily used as experimental controls for either primary or secondary antibodies. Antigen retrieval buffer, depends on specific antigen retrieval method: for HIAR e.g. There are several alkaline phosphatase inhibitors available, including levamisole hydrochloride and tetramisole hydrochloride. An increase in signal as the concentration decreases suggests that antibody concentration is too high. Many of the options listed above can then be tested to identify the cause ofautofluorescence. Sample concentration can be determined by comparison to a standard curve generated with a with a positive control. HHS Vulnerability Disclosure, Help HIAR using either a microwave oven or a pressure cooker is a common approach as they typically have a higher success rate for restoring antigenicity than an enzymatic method. The best negative control, aside from pre-immune serum mentioned above, is to pre-adsorb the primary antibody with an excess of the purified target protein. IHC. A simple test to verify that the enzyme and substrate are reacting properly is to place a drop of the enzyme onto a piece of nitrocellulose and then immediately dip it into the prepared substrate. Use a counterstain for specific morphologies or structures to aid localization of your primary antibody. False positives and negatives can be due to the immunohistochemical method itself but also to a myriad of other factors including preparation and fixation (see Notes section). Cool slides for 15 minutes at room temperature. Additional notes on reducing high background staining, Additional notes on increasing staining intensity, Copyright 2006-2023 Thermo Fisher Scientific Inc. All rights reserved, Immunofluorescence Method for IHC Detection, How to Choose and Select a Secondary Antibody.