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Immunohistochemistry—IHC5 steps to publication-quality images |
What is immunohistochemistry?
Immunohistochemistry (IHC) is a technique that uses an antibody to bind a specific antigen in a tissue section and is visualized with a fluorophore or colored substrate. Locations of stained proteins helps us understand cell types and their functions within a tissue. Basic IHC staining typically requires sample preparation, antigen retrieval, blocking, target detection, and visualization. Here we summarize the basic steps for IHC and the tools available for obtaining publication-quality images.
5 Steps for great IHC images
Step 1: Prepare sample
Once a tissue sample is harvested, it needs to be properly prepared for microscopic analysis by IHC. This requires preservation of samples for short- or long-term use, sectioning, and mounting tissue slices onto slides prior to starting the tissue staining process. Freezing or paraffin embedding are two approaches used for preserving tissues. Each method offers advantages and disadvantages, some of which are highlighted here.
Frozen sample preparation
Tissue samples are subjected to snap freezing by immersing the specimen in a super-cooled liquid such as liquid nitrogen or submerging in dry ice. Frozen tissues are suitable for short-term storage at -80° C for up to one year. Frozen tissue slices are generated using a cryostat.
Freezing the tissue offers a few advantages which include:
- Rapid preservation of the tissue sample
- Elimination of the need for antigen unmasking
- A preferred method for detection of post-translational modifications such as phosphorylation
The main disadvantage to using frozen samples is the potential for the formation of ice crystals and tissue damage if the tissue is not frozen rapidly.
Paraffin-embedded sample preparation
Paraffin-embedded tissues, also known as formalin-fixed paraffin-embedded (FFPE) tissues, are advantageous for preserving tissue morphology and long-term storage for several years. This approach requires a few steps to remove most of the water in the specimen before infiltrating it with paraffin, a hydrophobic substance. These steps include:
- Tissue fixation by perfusion or immersion in a fixative such as formaldehyde solution for up to 24 hours
- Dehydration using increasing concentrations of ethanol
- Treatment with a clearing reagent such as xylene to remove alcohol from the tissue
- Embedding the tissue in paraffin; FFPE tissue slices are generated using a microtome
A major disadvantage of paraffin-embedding is that tissue fixation can mask epitopes, therefore, FFPE tissues require antigen retrieval to unmask epitopes in an antigen. Another disadvantage is that over-fixation can lead to high fluorescence background which could become problematic for fluorescence staining.
Sample preparation application data
Figure 1. Immunohistochemical staining of tissue prepared using frozen techniques. Rat brain (E13.5) tissue was frozen, sectioned, and subjected to immunohistochemical staining to detect SNAP25 protein expression (green fluorescence) using Invitrogen SNAP25 Polyclonal Antibody. Sections were also stained with an anti-beta tubulin 3/ TUJ1 antibody (red fluorescence) and nucleic acid counterstain, DAPI (blue fluorescence).
Figure 2. Immunohistochemical staining of formalin-fixed paraffin-embedding tissue. Human colon cancer tissue sections were prepared using formalin-fixed paraffin-embedded (FFPE) techniques. To expose target proteins, heat-induced epitope retrieval was performed using 10 mM sodium citrate (pH 6.0) buffer for 20 minutes at 95°C. Following antigen retrieval, tissues were blocked in 3% Thermo Scientific Blocker BSA (10X) in PBS for 30 minutes at room temperature and then probed with an Invitrogen Ezrin Monoclonal Antibody (3C12) at a dilution of 1:100 for 1 hour in a humidified chamber. Tissues were washed extensively with Thermo Scientific Triton X-100 Surfact-Amps Detergent Solution and endogenous peroxidase activity quenched with Thermo Scientific Peroxidase Suppressor for 30 minutes at room temperature. Detection was performed using Invitrogen Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP at a dilution of 1:500 followed by colorimetric detection using Thermo Scientific Metal Enhanced DAB Substrate Kit. Images were taken on a Zeiss Axiovision microscope at 40X magnification.
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Step 2: Retrieve antigen
Fixation is an important process for the preservation of tissue morphology. However, this process can lead to the cross-linking of proteins, thereby masking epitopes in the antigen and restricting antigen-antibody binding. Antigen retrieval methods aid in unmasking epitopes by breaking protein cross-links and significantly improving antibody access and binding to the protein of interest.
Note: Antigen retrieval is not required as often for cryosections or cultured cells. Antigen retrieval is also not needed for all antibodies. Unless the source of the primary antibody specifically states that it is needed, try to label without antigen retrieval first.
There are two different types of antigen retrieval methods: heat-induced epitope retrieval (HIER) and protease-induced epitope retrieval (PIER). The optimal retrieval method depends on several factors such as tissue type, duration and method of fixation, and primary antibody type. To obtain best results, it is recommended to start with HIER, which is a gentler epitope retrieval method, before trying PIER.
Heat-induced epitope retrieval (HIER)
In HIER, the most common approach, the tissue is treated with a retrieval buffer while applying heat in a microwave, double boiler, or a pressure cooker. The most used HIER buffers are sodium citrate (pH 6.0), EDTA (pH 8.0), and Tris-EDTA (pH 9.0). Retrieval conditions using HIER may need optimization since the results will be dependent on time, temperature, and pH of the buffer used, and different antigens and samples may differ between samples and antibodies.
Protease-induced epitope retrieval (PIER)
In PIER, antigenicity is restored by applying enzymes such as proteinase K, pepsin, and trypsin which can destroy the cross-linked bonds. Be cautious when using PIER as excessive enzymatic treatment may damage the tissue and tissue morphology. Retrieval conditions using PIER may need optimization since the results will be dependent on time, temperature, and enzyme type, and concentration.
Antigen retrieval application data
Figure 3. Immunohistochemical staining using heat-induced antigen retrieval. IHC was performed on cancer biopsies of deparaffinized human colon carcinoma tissues. To expose target proteins, samples were microwaved for 8 to 15 min in a solution of 10mM sodium citrate (pH6.0) buffer. Following this step, tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:1000 with Invitrogen Actin Monoclonal Antibody (mAbGEa) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with phosphate buffered saline with Poly 80 (PBST) and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and streptavidin-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.
Figure 4. Immunohistochemical staining using enzymatic antigen retrieval. Immunohistochemical analysis of a mouse lung transduced with the human placental alkaline phosphatase (hPLAP) expression vector and the paraffin-embedded sections were protease-treated for 12 minutes. The sections were then stained with Invitrogen Placental Alkaline Phosphatase Polyclonal Antibody at a dilution of 1:100, followed by detection with an alkaline phosphatase-conjugated secondary antibody and colorimetric substrate (red). Tissues were counterstained with hematoxylin (blue). Data courtesy of the Innovators Program.
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Step 3: Block
Protein blocking is a technique that uses protein-based components to bind to structures that would otherwise attract antibodies. To help mediate this, a protein blocking reagent must be used to bind to the other “non-target” proteins. Minimizing background staining and reducing false positive signals can be achieved by incubating tissue samples with specific blocking reagents that address each background source. Endogenous enzyme blocking, autofluorescence, and non-specific primary and secondary antibody binding are major contributing sources of background signal in tissues.
Note: For staining intracellular targets, you have the option of permeabilizing tissue sections prior to treatment with blocking reagents.
Endogenous enzyme blocking
Types of endogenous enzyme blocking include:
- Biotin blocking—if you perform a streptavidin-biotin amplification block endogenous biotin in the sample first. Biotin is found primarily in the mitochondria of cells.
- Peroxidase blocking—if you perform an HRP conjugate detection, such as tyramide signal amplification, you first must inactivate endogenous peroxidases with an H2O2 blocking step.
- Phosphatase blocking—if you perform a detection for an alkaline phosphatase conjugate (like BCIP/NBT), endogenous phosphatases must first be inactivated.
Autofluorescence
Autofluorescence is native fluorescent background that is caused either by endogenous proteins or chemical treatments. Autofluorescence is usually broad-spectrum and lowers the signal-to-background ratio leading to reduced sensitivity. To block the background, you can either use chemical treatments or amplification techniques.
Non-specific antibody binding
Non-specific antibody binding can be mediated using protein blocking reagents. These reagents bind to structures that would otherwise attract antibodies. Not all protein blockers work equally for all targets, please choose one that would work best for your research.
| Blocker type | Product name | Cat. No. |
|---|---|---|
| Non-specific antibody binding | BlockAid Blocking Solution | B10710 |
| ReadyProbes Mouse-on-Mouse IgG Blocking Solution (30X) | R37621 | |
| ReadyProbes 2.5% Normal Goat Serum (1X) | R37624 | |
| eBioscience IHC /ICC Blocking Buffer - Low Protein | 00-4953-54 | |
| eBioscience IHC/ICC Blocking Buffer - High Protein | 00-4952-54 |
Blocking application data
Figure 5. Colorimetric example of immunohistochemical staining using blocking techniques. Differentiated human colon adenocarcinoma tissue sections were prepared using formalin-fixed paraffin-embedded (FFPE) techniques. To expose target proteins, heat-induced epitope retrieval was performed using 10 mM sodium citrate buffer (pH 6.0) for 20 minutes at 95°C. Following antigen retrieval, tissues were blocked in 3% Thermo Scientific Blocker BSA (10X) in PBS for 30 minutes at room temperature and then probed with Invitrogen HSP90 alpha Polyclonal Antibody at a dilution of 1:100 for 1 hour in a humidified chamber (right panel). As a negative control, the primary antibody was eliminated from the staining procedure (left panel). Tissues were washed extensively with Thermo Scientific Triton X-100 Surfact-Amps Detergent Solution and endogenous peroxidase activity quenched with Thermo Scientific Peroxidase Suppressor for 30 minutes at room temperature. Detection was performed using Invitrogen Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP at a dilution of 1:250 followed by colorimetric detection using Thermo Scientific Metal Enhanced DAB Substrate Kit. Tissues were counterstained with hematoxylin. Images were taken on a Zeiss Axiovision microscope at 40X magnification (x1.6 Optovar).
Figure 6. Fluorescence example of immunohistochemical staining using blocking techniques. IHC analysis of ACTA2 was performed using formalin-fixed paraffin-embedded human ovarian carcinoma tissue sections. To expose the target protein, heat-induced epitope retrieval was performed on de-paraffinized sections using eBioscience IHC Antigen Retrieval Solution - High pH (10X) diluted to 1X solution in water in a decloaking chamber at 110°C for 15 minutes. Following antigen retrieval, the sections were blocked with 2% normal goat serum in 1X PBS for 45 minutes at room temperature and then probed with or without eBioscience Alpha-Smooth Muscle Actin Monoclonal Antibody (1A4) at 1:100 dilution in 0.1% normal goat serum overnight at 4°C in a humidified chamber. Detection was performed using Invitrogen Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 at a dilution of 1:2,000 in 0.1% normal goat serum for 45 minutes at room temperature. Invitrogen ReadyProbes Tissue Autofluorescence Quenching Kit was used to quench autofluorescence from the tissues. Nuclei were stained with Invitrogen DAPI and the sections were mounted using Invitrogen ProLong Glass Antifade Mountant. The images were captured on Invitrogen EVOS M7000 Imaging System (Cat. No. AMF7000) at 20X magnification.
Step 3: Detect
Targets are detected by using fluorescence or chromogenic conjugated antibodies via direct or indirect staining methods.
Fluorescent dyes such as Alexa Fluor or Alexa Fluor Plus conjugates are among the most used dyes for immunofluorescence staining. Tyramide signal amplification can be used for enhanced and more sensitive fluorescent detection of low-abundance and hard-to-detect targets.
Horseradish peroxidase (HRP) or alkaline phosphatase (AP) are commonly used enzyme conjugates for chromogenic staining.
Note: Chemical or fluorescent counterstains can be used to complement antibody staining to visualize discrete cellular compartments.
Indirect staining
For indirect staining of tissues, a combination of unconjugated primary antibodies and fluorescent- or enzyme-conjugated secondary antibodies are used.
Note: To ensure a successful staining, always choose a primary antibody that has been tested and verified for use in IHC application.
Figure 7. Schematic comparing direct and indirect detection strategies.
Indirect staining protocols:
Direct staining
For direct staining of tissues, the need for secondary antibodies is bypassed. Instead, tissues are directly stained with a conjugated format of the primary antibody.
Note: To ensure a successful staining, always choose a primary antibody that has been tested and verified for use in IHC application.
Direct staining protocols:
Detection application data
Figure 8. Immunohistochemical analysis of β-3 tubulin (green) and PAX6 (magenta) in human iPSC–derived forebrain organoids. At day 40, the human iPSC–derived organoids were fixed with 4% formaldehyde for 1 hr at room temperature and incubated in a 30% sucrose solution overnight at 4°C. The organoids were then embedded in OCT (optimal cutting temperature compound) and cryo-sectioned at 5 µm, permeabilized with 0.2% Triton X-100 detergent for 20 min and blocked with 10% donkey serum in PBS for 30 min at room temperature. Organoid slices were incubated with a 1:500 dilution of Invitrogen anti–β-3 tubulin mouse monoclonal antibody (clone 2G10) and Invitrogen anti-PAX6 rabbit polyclonal antibody in blocking buffer overnight at 4°C, then stained with a 1:1,000 dilution of Invitrogen Alexa Fluor 488 donkey anti–mouse IgG ReadyProbes secondary antibody (green) and Invitrogen Alexa Fluor 568 donkey anti–rabbit IgG secondary antibody (magenta), as well as DAPI (blue), in blocking solution at room temperature for 1 hr. Images were taken on a Nikon Inverted Eclipse Ti-E Microscope at 20x magnification. Scale bar: 50 µm. Reprinted with permission from Zhexing Wen, Assistant Professor, Emory University School of Medicine, Atlanta, Georgia, USA.
Figure 9. Colorimetric detection of skeletal muscle tissue targets. Immunohistochemistry was performed on human skeletal muscle tissue prepared using formalin-fixed paraffin-embedded (FFPE) techniques. To expose target proteins, heat-induced antigen retrieval was performed using 10 mM sodium citrate buffer (pH 6.0) for 10 minutes using a microwave. Following antigen retrieval, tissues were blocked in 3% BSA-PBS for 30 minutes and then probed with (right panel) or without (left panel) Invitrogen MUSK Polyclonal Antibody, Rabbit at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively with Thermo Scientific Triton X-100 Surfact-Amps Detergent Solution and endogenous peroxidase activity quenched with Thermo Scientific Peroxidase Suppressor for 30 minutes at room temperature. Detection was performed using Invitrogen Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP followed by colorimetric detection using Thermo Scientific Metal Enhanced DAB Substrate Kit. Tissues were counterstained with hematoxylin.
Immunohistochemistry of formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Analysis was performed to compare SuperBoost (A) EverRed & (B) EverBlue staining in FFPE sections of human tonsil tissue compared (C) DAB staining. To expose target proteins, heat-induced epitope retrieval (HIER) was performed using 10 mM sodium citrate (pH 6.0), followed by heating in a pressure cooker for 20 minutes. After HIER, tissues were incubated in 3% H2O2 for 10 minutes at room temperature, blocked with blocking reagent, and then probed overnight at 4°C in a humid environment with an Invitrogen Ki-67 monoclonal antibody (Cat. No. MA5-14520), diluted 1:20 in PBS/3% (w/v) BSA. Tissues were washed extensively in PBS buffer containing 0.05% (v/v) Tween-20 (PBST). Detection was performed with the SuperBoost (A) EverRed Goat anti-Rabbit IgG (Cat. No. E40967), (B) EverBlue Goat anti-Rabbit IgG (Cat. No. E40968), or (C) DAB using SuperBoost Goat anti-Rabbit Poly HRP IgG (Cat. No. B40962). The sections were dehydrated with ethanol and xylene prior to mounting. Images were taken using an EVOS M7000 Imaging System (Cat. No. AMF7000) with 4x objective.
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Step 5: Visualize
In the final IHC step, the samples can be visualized. Stained tissue slides are commonly visualized by light or fluorescence microscopy using either a widefield or confocal imaging modality. For automated acquisition and analysis, several high content systems are available that can initially identify the tissue section using low magnification. Then, the area can be re-scanned using higher magnification objectives.
Mounting media
Before taking images it’s important to choose an appropriate mounting media to maintain good sample condition and address any photo bleaching that may occur. Here are a few scenarios that may occur when running IHC:
- If using fluorescent dyes, it is important to use an antifade mounting media that will slow any photobleaching of the dyes.
- If imaging your sample right away and then discarding the slide it would be best to use a non-curing mountant.
- If slides will be archived a curing mountant should be selected. A curing mountant firms to a hard gel and allows for long-term storage.
Visualization application data
Figure 10. Fluorescence visualization of immunohistochemically stained sample.Rat duodenal cryosections were stained with anti-histone H3 primary antibody followed by secondary detection using Invitrogen Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (green) using standard IHC protocols. The tissue sections were then stained with Invitrogen CellMask Deep Red Actin Tracking Stain (magenta) at 1X concentration and Hoechst 34580 (blue) for 1 hr. The tissue sections were then mounted with Invitrogen ProLong Glass Antifade Mountant. Images were taken using an Invitrogen EVOS M7000 imaging system.
Figure 11. Histological data acquisition and analysis on the CellInsight CX7 HCA Platform. Tissue microarrays of a diffuse large B cell lymphoma of neck were stained with the blue nuclear counterstain hematoxylin, the red cytoplasmic counterstain eosin Y, and human anti–Ki-67 in conjunction with a horseradish peroxidase (HRP)–conjugated secondary antibody and diaminobenzidine (DAB), which forms an insoluble brown product. (A) A stained tissue microarray core sample was acquired as separate fields using blue, green, and red illumination, a 40x objective, and tiling to create the entire sample. (B) Images were color-encoded to represent the typical visual acquired with a color camera. (C) Images were then individually analyzed: objects detected (blue outlines) and Ki-67 staining (red spots) were measured, analyzed, and compared to other groups within the array.
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Learn more about the EVOS Imaging Systems
Learn more about High-Content Screening (HCS) and High-Content Analysis (HCA) Systems
Step 1: Prepare sample
Once a tissue sample is harvested, it needs to be properly prepared for microscopic analysis by IHC. This requires preservation of samples for short- or long-term use, sectioning, and mounting tissue slices onto slides prior to starting the tissue staining process. Freezing or paraffin embedding are two approaches used for preserving tissues. Each method offers advantages and disadvantages, some of which are highlighted here.
Frozen sample preparation
Tissue samples are subjected to snap freezing by immersing the specimen in a super-cooled liquid such as liquid nitrogen or submerging in dry ice. Frozen tissues are suitable for short-term storage at -80° C for up to one year. Frozen tissue slices are generated using a cryostat.
Freezing the tissue offers a few advantages which include:
- Rapid preservation of the tissue sample
- Elimination of the need for antigen unmasking
- A preferred method for detection of post-translational modifications such as phosphorylation
The main disadvantage to using frozen samples is the potential for the formation of ice crystals and tissue damage if the tissue is not frozen rapidly.
Paraffin-embedded sample preparation
Paraffin-embedded tissues, also known as formalin-fixed paraffin-embedded (FFPE) tissues, are advantageous for preserving tissue morphology and long-term storage for several years. This approach requires a few steps to remove most of the water in the specimen before infiltrating it with paraffin, a hydrophobic substance. These steps include:
- Tissue fixation by perfusion or immersion in a fixative such as formaldehyde solution for up to 24 hours
- Dehydration using increasing concentrations of ethanol
- Treatment with a clearing reagent such as xylene to remove alcohol from the tissue
- Embedding the tissue in paraffin; FFPE tissue slices are generated using a microtome
A major disadvantage of paraffin-embedding is that tissue fixation can mask epitopes, therefore, FFPE tissues require antigen retrieval to unmask epitopes in an antigen. Another disadvantage is that over-fixation can lead to high fluorescence background which could become problematic for fluorescence staining.
Sample preparation application data
Figure 1. Immunohistochemical staining of tissue prepared using frozen techniques. Rat brain (E13.5) tissue was frozen, sectioned, and subjected to immunohistochemical staining to detect SNAP25 protein expression (green fluorescence) using Invitrogen SNAP25 Polyclonal Antibody. Sections were also stained with an anti-beta tubulin 3/ TUJ1 antibody (red fluorescence) and nucleic acid counterstain, DAPI (blue fluorescence).
Figure 2. Immunohistochemical staining of formalin-fixed paraffin-embedding tissue. Human colon cancer tissue sections were prepared using formalin-fixed paraffin-embedded (FFPE) techniques. To expose target proteins, heat-induced epitope retrieval was performed using 10 mM sodium citrate (pH 6.0) buffer for 20 minutes at 95°C. Following antigen retrieval, tissues were blocked in 3% Thermo Scientific Blocker BSA (10X) in PBS for 30 minutes at room temperature and then probed with an Invitrogen Ezrin Monoclonal Antibody (3C12) at a dilution of 1:100 for 1 hour in a humidified chamber. Tissues were washed extensively with Thermo Scientific Triton X-100 Surfact-Amps Detergent Solution and endogenous peroxidase activity quenched with Thermo Scientific Peroxidase Suppressor for 30 minutes at room temperature. Detection was performed using Invitrogen Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP at a dilution of 1:500 followed by colorimetric detection using Thermo Scientific Metal Enhanced DAB Substrate Kit. Images were taken on a Zeiss Axiovision microscope at 40X magnification.
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Step 2: Retrieve antigen
Fixation is an important process for the preservation of tissue morphology. However, this process can lead to the cross-linking of proteins, thereby masking epitopes in the antigen and restricting antigen-antibody binding. Antigen retrieval methods aid in unmasking epitopes by breaking protein cross-links and significantly improving antibody access and binding to the protein of interest.
Note: Antigen retrieval is not required as often for cryosections or cultured cells. Antigen retrieval is also not needed for all antibodies. Unless the source of the primary antibody specifically states that it is needed, try to label without antigen retrieval first.
There are two different types of antigen retrieval methods: heat-induced epitope retrieval (HIER) and protease-induced epitope retrieval (PIER). The optimal retrieval method depends on several factors such as tissue type, duration and method of fixation, and primary antibody type. To obtain best results, it is recommended to start with HIER, which is a gentler epitope retrieval method, before trying PIER.
Heat-induced epitope retrieval (HIER)
In HIER, the most common approach, the tissue is treated with a retrieval buffer while applying heat in a microwave, double boiler, or a pressure cooker. The most used HIER buffers are sodium citrate (pH 6.0), EDTA (pH 8.0), and Tris-EDTA (pH 9.0). Retrieval conditions using HIER may need optimization since the results will be dependent on time, temperature, and pH of the buffer used, and different antigens and samples may differ between samples and antibodies.
Protease-induced epitope retrieval (PIER)
In PIER, antigenicity is restored by applying enzymes such as proteinase K, pepsin, and trypsin which can destroy the cross-linked bonds. Be cautious when using PIER as excessive enzymatic treatment may damage the tissue and tissue morphology. Retrieval conditions using PIER may need optimization since the results will be dependent on time, temperature, and enzyme type, and concentration.
Antigen retrieval application data
Figure 3. Immunohistochemical staining using heat-induced antigen retrieval. IHC was performed on cancer biopsies of deparaffinized human colon carcinoma tissues. To expose target proteins, samples were microwaved for 8 to 15 min in a solution of 10mM sodium citrate (pH6.0) buffer. Following this step, tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:1000 with Invitrogen Actin Monoclonal Antibody (mAbGEa) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with phosphate buffered saline with Poly 80 (PBST) and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and streptavidin-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.
Figure 4. Immunohistochemical staining using enzymatic antigen retrieval. Immunohistochemical analysis of a mouse lung transduced with the human placental alkaline phosphatase (hPLAP) expression vector and the paraffin-embedded sections were protease-treated for 12 minutes. The sections were then stained with Invitrogen Placental Alkaline Phosphatase Polyclonal Antibody at a dilution of 1:100, followed by detection with an alkaline phosphatase-conjugated secondary antibody and colorimetric substrate (red). Tissues were counterstained with hematoxylin (blue). Data courtesy of the Innovators Program.
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Step 3: Block
Protein blocking is a technique that uses protein-based components to bind to structures that would otherwise attract antibodies. To help mediate this, a protein blocking reagent must be used to bind to the other “non-target” proteins. Minimizing background staining and reducing false positive signals can be achieved by incubating tissue samples with specific blocking reagents that address each background source. Endogenous enzyme blocking, autofluorescence, and non-specific primary and secondary antibody binding are major contributing sources of background signal in tissues.
Note: For staining intracellular targets, you have the option of permeabilizing tissue sections prior to treatment with blocking reagents.
Endogenous enzyme blocking
Types of endogenous enzyme blocking include:
- Biotin blocking—if you perform a streptavidin-biotin amplification block endogenous biotin in the sample first. Biotin is found primarily in the mitochondria of cells.
- Peroxidase blocking—if you perform an HRP conjugate detection, such as tyramide signal amplification, you first must inactivate endogenous peroxidases with an H2O2 blocking step.
- Phosphatase blocking—if you perform a detection for an alkaline phosphatase conjugate (like BCIP/NBT), endogenous phosphatases must first be inactivated.
Autofluorescence
Autofluorescence is native fluorescent background that is caused either by endogenous proteins or chemical treatments. Autofluorescence is usually broad-spectrum and lowers the signal-to-background ratio leading to reduced sensitivity. To block the background, you can either use chemical treatments or amplification techniques.
Non-specific antibody binding
Non-specific antibody binding can be mediated using protein blocking reagents. These reagents bind to structures that would otherwise attract antibodies. Not all protein blockers work equally for all targets, please choose one that would work best for your research.
| Blocker type | Product name | Cat. No. |
|---|---|---|
| Non-specific antibody binding | BlockAid Blocking Solution | B10710 |
| ReadyProbes Mouse-on-Mouse IgG Blocking Solution (30X) | R37621 | |
| ReadyProbes 2.5% Normal Goat Serum (1X) | R37624 | |
| eBioscience IHC /ICC Blocking Buffer - Low Protein | 00-4953-54 | |
| eBioscience IHC/ICC Blocking Buffer - High Protein | 00-4952-54 |
Blocking application data
Figure 5. Colorimetric example of immunohistochemical staining using blocking techniques. Differentiated human colon adenocarcinoma tissue sections were prepared using formalin-fixed paraffin-embedded (FFPE) techniques. To expose target proteins, heat-induced epitope retrieval was performed using 10 mM sodium citrate buffer (pH 6.0) for 20 minutes at 95°C. Following antigen retrieval, tissues were blocked in 3% Thermo Scientific Blocker BSA (10X) in PBS for 30 minutes at room temperature and then probed with Invitrogen HSP90 alpha Polyclonal Antibody at a dilution of 1:100 for 1 hour in a humidified chamber (right panel). As a negative control, the primary antibody was eliminated from the staining procedure (left panel). Tissues were washed extensively with Thermo Scientific Triton X-100 Surfact-Amps Detergent Solution and endogenous peroxidase activity quenched with Thermo Scientific Peroxidase Suppressor for 30 minutes at room temperature. Detection was performed using Invitrogen Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP at a dilution of 1:250 followed by colorimetric detection using Thermo Scientific Metal Enhanced DAB Substrate Kit. Tissues were counterstained with hematoxylin. Images were taken on a Zeiss Axiovision microscope at 40X magnification (x1.6 Optovar).
Figure 6. Fluorescence example of immunohistochemical staining using blocking techniques. IHC analysis of ACTA2 was performed using formalin-fixed paraffin-embedded human ovarian carcinoma tissue sections. To expose the target protein, heat-induced epitope retrieval was performed on de-paraffinized sections using eBioscience IHC Antigen Retrieval Solution - High pH (10X) diluted to 1X solution in water in a decloaking chamber at 110°C for 15 minutes. Following antigen retrieval, the sections were blocked with 2% normal goat serum in 1X PBS for 45 minutes at room temperature and then probed with or without eBioscience Alpha-Smooth Muscle Actin Monoclonal Antibody (1A4) at 1:100 dilution in 0.1% normal goat serum overnight at 4°C in a humidified chamber. Detection was performed using Invitrogen Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 at a dilution of 1:2,000 in 0.1% normal goat serum for 45 minutes at room temperature. Invitrogen ReadyProbes Tissue Autofluorescence Quenching Kit was used to quench autofluorescence from the tissues. Nuclei were stained with Invitrogen DAPI and the sections were mounted using Invitrogen ProLong Glass Antifade Mountant. The images were captured on Invitrogen EVOS M7000 Imaging System (Cat. No. AMF7000) at 20X magnification.
Step 3: Detect
Targets are detected by using fluorescence or chromogenic conjugated antibodies via direct or indirect staining methods.
Fluorescent dyes such as Alexa Fluor or Alexa Fluor Plus conjugates are among the most used dyes for immunofluorescence staining. Tyramide signal amplification can be used for enhanced and more sensitive fluorescent detection of low-abundance and hard-to-detect targets.
Horseradish peroxidase (HRP) or alkaline phosphatase (AP) are commonly used enzyme conjugates for chromogenic staining.
Note: Chemical or fluorescent counterstains can be used to complement antibody staining to visualize discrete cellular compartments.
Indirect staining
For indirect staining of tissues, a combination of unconjugated primary antibodies and fluorescent- or enzyme-conjugated secondary antibodies are used.
Note: To ensure a successful staining, always choose a primary antibody that has been tested and verified for use in IHC application.
Figure 7. Schematic comparing direct and indirect detection strategies.
Indirect staining protocols:
Direct staining
For direct staining of tissues, the need for secondary antibodies is bypassed. Instead, tissues are directly stained with a conjugated format of the primary antibody.
Note: To ensure a successful staining, always choose a primary antibody that has been tested and verified for use in IHC application.
Direct staining protocols:
Detection application data
Figure 8. Immunohistochemical analysis of β-3 tubulin (green) and PAX6 (magenta) in human iPSC–derived forebrain organoids. At day 40, the human iPSC–derived organoids were fixed with 4% formaldehyde for 1 hr at room temperature and incubated in a 30% sucrose solution overnight at 4°C. The organoids were then embedded in OCT (optimal cutting temperature compound) and cryo-sectioned at 5 µm, permeabilized with 0.2% Triton X-100 detergent for 20 min and blocked with 10% donkey serum in PBS for 30 min at room temperature. Organoid slices were incubated with a 1:500 dilution of Invitrogen anti–β-3 tubulin mouse monoclonal antibody (clone 2G10) and Invitrogen anti-PAX6 rabbit polyclonal antibody in blocking buffer overnight at 4°C, then stained with a 1:1,000 dilution of Invitrogen Alexa Fluor 488 donkey anti–mouse IgG ReadyProbes secondary antibody (green) and Invitrogen Alexa Fluor 568 donkey anti–rabbit IgG secondary antibody (magenta), as well as DAPI (blue), in blocking solution at room temperature for 1 hr. Images were taken on a Nikon Inverted Eclipse Ti-E Microscope at 20x magnification. Scale bar: 50 µm. Reprinted with permission from Zhexing Wen, Assistant Professor, Emory University School of Medicine, Atlanta, Georgia, USA.
Figure 9. Colorimetric detection of skeletal muscle tissue targets. Immunohistochemistry was performed on human skeletal muscle tissue prepared using formalin-fixed paraffin-embedded (FFPE) techniques. To expose target proteins, heat-induced antigen retrieval was performed using 10 mM sodium citrate buffer (pH 6.0) for 10 minutes using a microwave. Following antigen retrieval, tissues were blocked in 3% BSA-PBS for 30 minutes and then probed with (right panel) or without (left panel) Invitrogen MUSK Polyclonal Antibody, Rabbit at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively with Thermo Scientific Triton X-100 Surfact-Amps Detergent Solution and endogenous peroxidase activity quenched with Thermo Scientific Peroxidase Suppressor for 30 minutes at room temperature. Detection was performed using Invitrogen Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP followed by colorimetric detection using Thermo Scientific Metal Enhanced DAB Substrate Kit. Tissues were counterstained with hematoxylin.
Immunohistochemistry of formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Analysis was performed to compare SuperBoost (A) EverRed & (B) EverBlue staining in FFPE sections of human tonsil tissue compared (C) DAB staining. To expose target proteins, heat-induced epitope retrieval (HIER) was performed using 10 mM sodium citrate (pH 6.0), followed by heating in a pressure cooker for 20 minutes. After HIER, tissues were incubated in 3% H2O2 for 10 minutes at room temperature, blocked with blocking reagent, and then probed overnight at 4°C in a humid environment with an Invitrogen Ki-67 monoclonal antibody (Cat. No. MA5-14520), diluted 1:20 in PBS/3% (w/v) BSA. Tissues were washed extensively in PBS buffer containing 0.05% (v/v) Tween-20 (PBST). Detection was performed with the SuperBoost (A) EverRed Goat anti-Rabbit IgG (Cat. No. E40967), (B) EverBlue Goat anti-Rabbit IgG (Cat. No. E40968), or (C) DAB using SuperBoost Goat anti-Rabbit Poly HRP IgG (Cat. No. B40962). The sections were dehydrated with ethanol and xylene prior to mounting. Images were taken using an EVOS M7000 Imaging System (Cat. No. AMF7000) with 4x objective.
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Step 5: Visualize
In the final IHC step, the samples can be visualized. Stained tissue slides are commonly visualized by light or fluorescence microscopy using either a widefield or confocal imaging modality. For automated acquisition and analysis, several high content systems are available that can initially identify the tissue section using low magnification. Then, the area can be re-scanned using higher magnification objectives.
Mounting media
Before taking images it’s important to choose an appropriate mounting media to maintain good sample condition and address any photo bleaching that may occur. Here are a few scenarios that may occur when running IHC:
- If using fluorescent dyes, it is important to use an antifade mounting media that will slow any photobleaching of the dyes.
- If imaging your sample right away and then discarding the slide it would be best to use a non-curing mountant.
- If slides will be archived a curing mountant should be selected. A curing mountant firms to a hard gel and allows for long-term storage.
Visualization application data
Figure 10. Fluorescence visualization of immunohistochemically stained sample.Rat duodenal cryosections were stained with anti-histone H3 primary antibody followed by secondary detection using Invitrogen Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (green) using standard IHC protocols. The tissue sections were then stained with Invitrogen CellMask Deep Red Actin Tracking Stain (magenta) at 1X concentration and Hoechst 34580 (blue) for 1 hr. The tissue sections were then mounted with Invitrogen ProLong Glass Antifade Mountant. Images were taken using an Invitrogen EVOS M7000 imaging system.
Figure 11. Histological data acquisition and analysis on the CellInsight CX7 HCA Platform. Tissue microarrays of a diffuse large B cell lymphoma of neck were stained with the blue nuclear counterstain hematoxylin, the red cytoplasmic counterstain eosin Y, and human anti–Ki-67 in conjunction with a horseradish peroxidase (HRP)–conjugated secondary antibody and diaminobenzidine (DAB), which forms an insoluble brown product. (A) A stained tissue microarray core sample was acquired as separate fields using blue, green, and red illumination, a 40x objective, and tiling to create the entire sample. (B) Images were color-encoded to represent the typical visual acquired with a color camera. (C) Images were then individually analyzed: objects detected (blue outlines) and Ki-67 staining (red spots) were measured, analyzed, and compared to other groups within the array.
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Learn more about the EVOS Imaging Systems
Learn more about High-Content Screening (HCS) and High-Content Analysis (HCA) Systems
IHC tips and tricks videos
Webinars
New on-demand webinar: Tips and tricks for successful multiplexing IHC
Expanding to include more markers in an IHC experiment, requires in-depth knowledge about sample preparation, fluorescent dyes, and microscopes. This webinar will elucidate how to select markers and fluorophore dyes to a simple IHC experiment or start a larger spatial imaging panel. We will discuss how to prevent sample autofluorescence, non-specific antibody labeling, fluorescent bleed-through and spectral overlap.
Webinar: Five steps for publication-quality immunohistochemistry imaging
Immunohistochemistry protocols, which utilize antibodies to visualize proteins in tissue sections, have many steps that need optimization to prevent nonspecific background effects, artifacts, or inadequate detection by dyes.
Resources
Overview of Immunohistochemistry
Read an in-depth article on the principles of immunohistochemistry and the methods used to get great results.
IHC Troubleshooting Guide
Having issues with your protocol? Check out this guide to help resolve some of the issues you might be having. Topics include strong background staining, weak target staining, and autofluorescence.
Antibodies for Immunohistochemistry (IHC)
Ready to choose your antibodies for detection? This article outlines the considerations to keep in mind when selecting the best antibodies for your research.
IHC Immunodetection
Read a comprehensive article to learn more about the cornerstone of immunohistochemistry – antibody-mediated target antigen detection/staining.
IHC Counterstains
Looking to provide contrast that helps your staining of the target antigen stand out? Read this article to learn more about chemical and fluorescent counterstains.
Stylesheet for Classic Wide Template adjustments
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