Manual Techniques in Immunocytochemistry

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The most challenging aspect of these techniques is determining the experimental conditions necessary to generate a strong and specific signal for each antigen of interest. For example, detection of an abundant protein in cultured cells may require a short fixation period, minimal blocking, and may be compatible with direct visualization using a fluorochrome-conjugated primary antibody. In contrast, detection of a phosphorylation-dependent epitope in a section of frozen tissue may require antigen retrieval and be dependent on amplified chromogenic visualization.

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Designing a Successful IHC/ICC Experiment

Skip to main content. Chromogenic detection by ICC was used to visualize IFN-gamma in human peripheral blood mononuclear cells stimulated with calcium ionomycin 0. Enzymatic detection is therefore more appropriate for tissue sections. Cytological preparations and frozen sections are commonly enough exposed to formaldehyde for long enough to exacerbate autofluorescence, and cytological preparations often do not possess such naturally fluorescent components.

Our detection system is now incubated for 1 h and we'll wash it off with a buffer rinse. If we're using an ABC system, it's at this stage that it will be incubated on the specimen. Make this up according to the manufacturer's instructions. The ABC system is used to provide as Avidin alone, with a Biotin label complex in a separate container. End users must mix both accordingly, and wait for at least 30 min for complex formation before adding it to the specimen, or it would not function to its full capability as a detection reagent.

However, if you are not using an ABC system, you'd skip step six and seven and proceed directly to step eight.

Immunohistochemistry vs Immunocytochemistry

If you are using an enzyme reporter label, you are now ready to add the chromogen. Reporter labels that are enzymatic in nature, produce a stable colored precipitate for the site of primary antibody binding when exposed to a suitable chromogen. Amongst several, the two most popular enzyme labels for immunochemistry are horseradish peroxidase and alkaline phosphatase. As you can see from the table, the different enzyme and chromogen combinations produce a different color precipitate at the site of antibody binding. Also note that some of the precipitators are alcohol-soluble which has an important significance when mounting, which I will come on to later.

Ensure that all chromogens are made up according to the manufacturer's instructions, and incubate them on a specimen according to manufacturer's guidelines. Typically for DAB, 10 min at room temperature generally gives good results. Always observe chromogen expiry dates as storage conditions. HRP and hydrogen peroxide form a complex in the presence of DAB chromogen with HRP catalyzing the breakdown of hydrogen peroxide into water and oxygen.

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DAB is oxidized during this process and provides electrons to drive the reaction. Hydrogen peroxide, therefore, plays a key role in this reaction and since it quickly breaks down into water and oxygen at room temperature, DAB kits that have passed their expiry date may be ineffective. Appropriate COSHH guidelines should be observed regarding the storage, use, handling and disposal of any laboratory agents.

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If using an alkaline phosphatase reporter label add 0. Levamisole quenched endogenous phosphatase activity, reducing unwanted background staining, although not in placenta or small intestine. Don't worry, the phosphatase reporter label itself will not be affected by the Levamisole. After washing off the chromogen and running domestic supply water, we can now apply a suitable counterstain.

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Counterstains add color-contrasted the cells or tissues by staining certain cellular structures, thus helping to define the localization of the immunostaining. They can be tinctoral or fluorescent in nature, complementive in section system used to visualize a primary antibody. For enzyme detection systems, tinctoral and nuclear counterstains are commonly used. I'll talk more about fluorescent counterstains in the second section of this webinar. The most common nuclear counterstaining used when employing an enzyme chromogen detection system, is Hemotoxylin.

Immunohistochemistry (IHC) & Antigen/Epitope Protein Retrieval - Principle, Technique and Protocol

Hemotoxylins are available in numerous formulations identified by the type of mordant used, and whether they stain progressively or regressively. Hemotoxylin stains cell nuclei various shades of purple or blue, according to the type used. Mordants are iron salts which combine with hematin, creating a positively charged dye-mordant complex with the ability to bind an ionic chromotin. Alum or aluminium mordant hemotoxylins can be used progressively or regressively. Progressive hemotoxylins, for example, are Mayer's, Gill's or Carazzi's can be applied to tissues or cells until the desired degree of nuclear staining is observed.

This process is called differentiation. This makes progressive hemotoxylins simpler to use and regressive due to the emission of the differentiation step, and the subsequence compatibility with alcohol-soluble enzymes, substrate end products, such as that produced by the reaction of HRP and AEC.

All hemotoxylins, both progressive and regressive, are blue to a desired level of staining has been achieved. Running domestic water, supply water is commonly used for this purpose.

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It usually has sufficient alkalinity to achieve this, especially in hard water areas. In soft water areas, an alkaline solution can be used for bluing such as 0. Other commonly used tinctoral nuclear counterstains are light green, fast red, toluidine blue and methylene blue; then in nuclei the green, red or blue, respectively. One important consideration when using a nuclear tinctoral counterstain, is not to make the staining too intense if you are demonstrating a nuclear antigen, since the counterstain can potentially mask a positive signal from the detection system.

Let's now discuss specimen preparation for microscopic analysis. It is essential to prepare the immunostain specimen in order to preserve it while being imaged during long-term storage, and to enhance image quality. This typically involves placing a glass coverslip over the specimen, securing it in place with a suitable adhesive known as mounting media. Mounting media can be either aqueous, suitable for both fluorescent and enzymatic labels, or organic suitable only for enzymatic.

Organic mounting medias tend to set hard, allowing the glass coverslip to remain securely in place. Refractive indexes are better with organic mounting media, such as DPX giving a much sharper, crisper image down the microscope. However, ensure that the color precipitate forms from the reaction of the enzymatic label with a substrate is compatible with organic mounting media. For example, the reaction of peroxidase and AEC is alcohol soluble, so will disappear during the dehydrating and cleaning process, if an organic mounting media is used.


When using a fluorescent label, it is advisable to microscopically observe the mounting media on a blank coverslip to ensure that it does not produce any autofluorescence. I'm happy to say that our immunostaining specimen is now ready to be analyzed down the microscope. Microscopic analysis is a complete webinar on its own, especially to go into how to setup a microscope optimum illumination compared with light microscopy and fluorescence; and so I won't be going into that right now.


Let's now think about multiple staining. The purpose of immunostaining using multiple reporter labels, is to simultaneously visualize the cellular localization of two or more antigens in the same cell or tissue section. A different reporter label is used for each antigen in order to distinguish them apart. Antigens may be co-localized, meaning within the same cellular compartment of any given cell, or separate.

When two or more antigens are co-localized, the color of the separate reporter labels will mix to produce a new color. Both fluorescent and enzyme reporter labels can be used, but these need to be carefully selected to ensure that they do not interfere with each other, and that they are easily interdistinguishable. Similarly, the detection system reagents must not cross-react or interfere with each other in any way.

This webinar will concentrate on fluorescence multi-staining, that way there's always the potential for another separate webinar for enzymatic later. Before we go on, we will first consider how fluorescence works. A fluorescent molecule has the ability to absorb light of a specific wavelength and to re-emit light at a longer wavelength. It loses energy in order to achieve this by interacting with its environment prior to the emission of fluorescence; a phenomenon called internal conversion, which is the loss of energy in the absence of light emission.

Electrons can exist either at a ground state or resting state called S0, or in excited states of higher energy called S1 and S2. At each of these electronic states the fluorochromes can exist in a number of vibrational levels called 0, 1 and 2. There is not enough energy at room temperature to populate the excited electronic states of S1 and S2, or the higher vibrational levels of the ground state.

Therefore, absorption generally occurs from molecules in the lowest vibrational energy state, and only in the presence of light. Following absorption, a fluorochrome is usually excited to a higher vibrational level and be the S1 or S2 before relaxing to the lowest vibrational level of S1, thus completing the process of internal conversion. This combined with the emission of a photon results in fluorescence.

A fluorochrome can repeat the excitation emission cycle many times before excitation bleaches the fluorescent signal, otherwise known as photobleaching. However, some fluorophores photobleach more readily than others. FITC, for example, can repeat the excitation emission process approximately 30, times before photobleaching occurs.