The following video shows immunohistochemistry (IHC) with paraffin sections, which reveal the tissue’s molecular architecture by using a wide range of stains and antibodies.
Sections embedded with paraffin are initially dewaxed to substitute the wax with water. This is done because most of the staining solutions are aqueous-based ones. After dewaxing, an antigen-retrieval method is performed which reveals antigens masked during the fixation procedure. Then, based on the type of antibodies to be used, a set of blocking steps occur that help limit non-specific interactions between the block endogenous enzymes and the antibody that can lead to false positives.
Blocking is followed by incubating the sections in antibodies; this incubation can be either in directly conjugated primaries or can include sequential incubations in primary as well as secondary antibodies (enzyme or fluorescent-linked).
If an enzyme-linked secondary is used, a further incubation in substrate will be required, otherwise the sections can be mounted and imaged.
Paraffin and Frozen Sections
A pipette can be used to manually apply reagents, or else this procedure can be adapted for semi-automated and automated systems, if these are available. Incubations are performed in a humidified chamber to make sure that the tissue does not dry out or otherwise this will lead to background staining and non-specific binding.
A shallow plastic box with wet tissue paper in the bottom and sealed lid serves as a sufficient chamber. To ensure that the reagents do not drain off, the slides are kept off the paper and laid flat.
Next, a plastic serological pipette is cut into lengths to fit the specified incubation chamber and then glued together in pairs to the chamber base, with each pair placed about 4 cm apart. This gives a raised and level surface, allowing the slides to rest on, away from the wet tissue paper.
Dilutions of both primary and secondary antibodies are either listed on the datasheets or established by testing a series of dilutions. Dilutions should be properly adjusted from the results acquired. Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the most common enzymes used for enzymatic methods. Several chromogens are used with these enzymes.
Save Time with Validated IHC Detection Kits and Reagents
Abcam’s pre-optimized IHC detection kits are recommended to complement the primary antibody and also to obtain rapid results:
Labeled Streptavidin-Biotin LSAB ABC kits are analogous to conventional avidin ABC kits, but they possess less background and greater sensitivity
Micro-polymer EXPOSE kits provide the best sensitivity, do not include any biotin blocking step and lower background
Abcam’s Mouse-on-Mouse kit available for utilizing mouse antibodies on mouse tissue
Dedicated kits available for triple-staining and double-staining
Users can also develop their own ABC method. For this, Abcam recommends its biotin and micro-polymer conjugated secondary antibodies and also proposes its range of normal serums for the blocking process.
Antigen retrieval should be carried out before starting with immunostaining, if required.
If a HRP conjugate for detection is being used, endogenous peroxidase can be blocked but it is recommended to wait until the incubation of the primary antibody.
H 2O 2 reduces background staining by suppressing the activity of endogenous peroxidase. The presence of endogenous peroxidases should be checked and this can be done by incubating a tissue slide following re-hydration in DAB solution. If section areas appear brown under the microscope, staining can be reduced through a blocking step.
Peroxide can modify certain epitopes, leading to reduced antibody-antigen binding. This problem can be prevented by incubating the sections with peroxide following the primary incubation.
Peroxide can either be diluted in water or TBS. Methanol proves handy for peroxidase-rich tissues or blood smears; tissue damage promoted by the reaction in aqueous solutions may be reduced by peroxide diluted in methanol.
For other types of tissues, peroxide can be diluted in either water or TBS. Restricted binding of certain antibody-antigen pairs, especially cell surface proteins, has been noted following the incubation of peroxide/methanol. If AP or fluorescent detection is used, peroxidase quenching can be omitted because it only applies to HRP conjugates.
The slides are washed for 2 x 5 minutes in TBS containing 0.025% Triton X-100 with gentle agitation.
0.025% Triton X-100 present in the TBS reduces surface tension, enabling the reagents to easily cover the entire tissue section. Triton X-100 is also believed to reduce non-specific binding and dissolve Fc receptors. To obtain a cleaner background, Abcam recommends using TBS over PBS.
At room temperature, 10% of normal serum is blocked with 1% of BSA in TBS for 2 hours.
It is likely that the secondary antibody may cross react with endogenous immunoglobulins within the tissue. To reduce this, the tissue can be pre-treated with normal serum from the species from where the secondary was raised.
Fc receptor binding of primary and secondary antibodies can also be prevented by using normal serum prior to the application of the primary antibody. Non-specific binding induced by hydrophobic interactions is reduced by including BSA.
The slides are drained for several seconds (not rinsed) and wiped around the sections with tissue paper.
Primary antibody diluted in TBS is applied with 1% BSA.
The primary antibody is diluted either according to the manufacturer’s recommendations or to an earlier optimized dilution. Majority of antibodies will be employed in IHC-P at 0.5 to 10 μg/mL concentration.
It is essential to raise the primary antibody in a species that differs from the tissue being stained. For instance, if a mouse tissue is available and the primary antibody is raised in a mouse, an anti-mouse IgG secondary antibody will lead to high background by binding to all the endogenous IgG in the mouse tissue. Abcam’s mouse-on-mouse protocol describes the use of mouse monoclonals on mouse tissue.
Overnight incubation at 4 °C is observed.
With overnight incubation, antibodies of lower affinity or titer can be used which allows more time for them to bind. Irrespective of the antibody's affinity or titer for its target, binding does not occur once the tissue reaches a saturation point. Overnight incubation is done to make sure that this takes place.
The slides are rinsed for 2 x 5 minutes in TBS plus 0.025% Triton with mild agitation.
If an HRP conjugate is used for detection, the slides are incubated in 0.3% H
2O 2 in TBS for 15 minutes.
The colored product of the enzyme with the right chromogen is developed. The option depends on the desired colored end product, enzyme label and whether organic or aqueous mounting media is employed.
INT, Fast Red, AEC or any other aqueous chromogen can be dissolved by alcohol. An appropriate aqueous mounting media can be used but this should not be dehydrated and cleared Detection Step
For enzymatic detection (AP or HRP secondary conjugates)
Enzyme-conjugated secondary antibody is applied to the slide, then diluted in TBS with 1% BSA, and incubated at room temperature for 1 hour.
Developed with chromogen for 10 minutes at room temperature.
Washed in running tap water for 5 minutes.
Counterstained, if needed – Some commonly used counterstains are methyl green, nuclear fast red or hematoxylin (blue). When using fluorescent detection propidium iodide/PI (red) or DAPI (blue) can be used.
Dehydrated, cleared and mounted.
INT, Fast Red, AEC or any other aqueous chromogen can be dissolved by alcohol. A suitable aqueous mounting media can be used but this should not be dehydrated and cleared.
The sections developed should be dehydrated and cleared using New Fuchsin, DAB, Vega Red, TNBT, NBT, or other organic chromogen developed sections. This can be done by running the rehydration steps in Abcam’s deparaffinization protocol. The sections should be mounted in an appropriate organic mounting medium.
Compared to sections mounted in aqueous mounting media, those mounted in organic mounting media tend to have a better refractive index. This gives a sharper microscopic image when using an organic mounting medium.
For Fluorescent Detection
Fluorophore-conjugated secondary antibody is applied to the slide diluted in TBS with 1% BSA.
Slide is incubated at room temperature 1 hour.
These steps should only be performed in the dark to prevent photobleaching.
Slides are rinsed for 3 x 5 minutes with TBS.
Using compatible mounting medium, slides are mounted and a coverslip is added.
Immunostaining: Free-floating Sections
Materials and Reagents
8 mL methanol
20 mL 0.2M phosphate buffer
2 mL hydrogen peroxide
80 µL Triton-X100
Make up to 40 mL with ddH
2O Blocking buffer
0.1M Phosphate buffer
1% serum from secondary antibody host species
30–40 µm sections are cut on a freezing microtome and then collected into a multi-well or plate petri dishes containing 1-2 mL of 0.1M phosphate buffer (PBS).
Sections are stored at 4 °C for a week. For extended storage, azide is added and the multi well plate or petri dish is wrapped in cling film and preserved at 4 °C for 2 months. This should not be left too long as it would be difficult to mount the sections.
Endogenous peroxidases are deactivated by incubating in peroxidase block for about 15 minutes with gentle agitation.
Sections are washed (3 x 15 minutes) in 0.1M PBS/0.3% Triton.
Sections are then incubated in blocking buffer for a minimum of 1 hour with gentle agitation.
They are again washed (3 x 15 minutes) in 0.1M PBS/0.3% Triton.
The primary antibody is added and incubated overnight at 4 °C with gentle agitation.
Sections are washed (3 x 15 minutes) in 0.1M PBS/0.3% Triton.
Secondary antibody is added either for 2 hours at room temperature or left overnight at 4 °C with gentle agitation.
Sections are washed (3 x 15 minutes) in 0.1M PBS (without Triton).
They are briefly washed in 0.1M acetate buffer to eliminate PBS.
Acetate buffer should be freshly prepared.
Sections are placed in solution to promote DAB reaction, which is later monitored on microscope for (2 to 10 minutes).
Once the background is sufficiently high, the reaction is ceased by placing the sections in 0.1M acetate buffer.
Sections are washed (3 x 15 minutes) in 0.1M PBS.
Sections can be stored in a cold room for 2 days until they are mounted and coverslipped.
They are air dried in fume hood and then placed (2 x 10 minutes) in histoclear.
They are mounted from the third 10 minutes histoclear incubation.
Coverslip using Ralmount. A suitable mounting medium is used for sections marked with fluorescent detection.
In order to obtain a stronger signal, a number of strategies have been devised to add more fluorophore or enzyme to the preferred target.
Avidin-biotin Complex (ABC)
This method leverages the avidin’s high affinity for biotin – an enzyme co-factor used in carboxylation reactions. For biotin, avidin has four binding sites with binding being basically irreversible.
In short, the primary antibody is bound to the target protein, followed by the binding of a biotinylated secondary antibody to the primary antibody. In another reaction, mixing the avidin and biotinylated enzyme in a ratio, which leaves certain binding sites on avidin unoccupied, leads to the formation of a complex. The resultant complex is subsequently incubated with the tissue section post the incubation of antibody.
Within the complex, the unoccupied biotin-binding sites bind to the biotinylated secondary antibody. As a result, more enzyme is attached to the target than is possible with a primary or enzyme-conjugated secondary antibody.
The parts of the avidin-biotin complex are now commercially available in kits and the complex can be employed with Abcam’s biotinylated antibodies. The endogenous biotin present in tissues can bind the avidin-biotin complex and result in background staining. Such tissues include brain, liver, kidney, testes, intestines, colon and prostate.
Labeled Streptavidin Biotin (LSAB)
Similar to ABC, this method employs the interaction of biotin and streptavidin (analogous to avidin in binding affinity). The primary antibody is followed by a biotinylated anti-lg secondary antibody, which in turn is followed by streptavidin conjugated to a fluorophore or enzyme. Since streptavidin is non-glycosylated (unlike avidin), it generates less non-specific background staining than that of avidin and therefore does not interact with carbohydrate-binding proteins, including lectins. For a comparison to ABS, where LSAB was shown to be as much as 4-8 times more sensitive, refer Giorno R (1984),
Diagn Immunol. 2:161–6. HRP Polymer
Novel polymer-enzyme-antibody products that contain a secondary antibody (for example, anti-mouse and/or rabbit IgG) bound to a polymer-enzyme complex, are being preferred over the avidin-biotin methods (LSAB and ABC). This is partly due to avoiding the problem of endogenous biotin.
EXPOSE detection kits meant for IHC staining protocol employ tiny linker-based detection modules that result in higher sensitivity, because these can enter the tissues better than the huge complexes employed in traditional polymer-based detection.
Tyramide Signal Enhancing (TSE)
The patented and licensed method, TSE (also called CSA or TSA) is an effective amplification procedure and is useful for detecting sparse antigens that are otherwise difficult to detect by other systems, and also for enhancing the results acquired with poorly-performing antibodies.
The TSE method depends on a peroxidase-catalyzed reaction to covalently bind to the tyramide part of tyramine-protein conjugates to the region around the target protein, following the application of a secondary-HRP conjugate and primary antibody.
Even if the slides are treated to remove the antibodies, it is not possible to wash off the covalently bound protein because the tyramide bond is covalent. When the fluorophore conjugate or antibody-enzyme is directed against the protein part of the tyramine-protein conjugate, a signal is achieved. The kits are costly and also multiple steps are involved that consume a lot of time.
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