Video Tutorials

Immunohistochemistry using the VECTASTAIN® ABC Kit

Chi, V., Chandy, K. G. Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs. J. Vis. Exp. (8), e308, doi:10.3791/308 (2007).

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

Western Blotting using the VECTASTAIN® ABC Kit

Gallagher, S., Chakavarti, D. Immunoblot Analysis. J. Vis. Exp.(16), e759, doi:10.3791/759 (2008).

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon. The immunoblotting technique has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive (1 ng of antigen can be detected). This unit provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Considerations for Designing Immunohistochemical Procedures

Part 1: Essential Choices for IHC

Quant Tag Kit for Biotin Quantitation

The QuantTag™ Biotin Kit (BDK-2000) from Vector Laboratories is designed to determine the amount of free biotin in solution or the number of biotins attached to nucleic acids, proteins, or other macromolecules. This kit can be used to determine accurately the labeling efficiency of biotin-labeled molecules.

Unlike conventional biotin quantitation methods like the HABA assay, no predigestion of protein or nucleic acids is required, saving time and increasing accuracy. The QuantTag™ Biotin Kit, more sensitive than the HABA assay, is able to detect less than 1 nmol of biotin. QuantTag™ Kit reagents chemically react with free or bound biotin producing a colored product that can be quantified using a spectrophotometer. The absorbance is measured in the visible spectrum allowing the use of plastic cuvettes or microtitre plates.

The QuantTag™ Biotin Kit is quick and easy to use, and the assay can be completed in 30 minutes. A biotin standard is included. The kit contains sufficient reagents to perform from 25 to 250 tests depending on assay size.

Dot Method

Part 2: Selecting Your IHC Reagents

Test Loading

Before paraffin sections can be stained in an immunohistochemical (IHC) procedure, your slides need to be deparaffinized and rehydrated. This tutorial will show you how to remove the wax (paraffin) with xylene or xylene substitute, then rehydrate your sections through a graded alcohol series.

Blocking of Endogenous Peroxidase Activity

Endogenous peroxidase (HRP) present in tissue sections can result in background when using an HRP detection system if this activity is not quenched. Methods for quenching this endogenous enzyme activity include treating with BLOXALL Endogenous Enzyme Blocking Solution or with various preparations of hydrogen peroxide. In this tutorial we demonstrate how to block endogenous peroxidase using a hydrogen peroxide incubation procedure.

VectaMount® Permanent Mounting Medium

VectaMount™ Mounting Medium (H-5000) from Vector Laboratories is an optically clear and odorless formula for permanently preserving histochemical stains or precipitable enzyme substrates in tissue sections or cell preparations. VectaMount™ Mounting Medium contains no toluene or xylene. It has a viscosity which provides for easy application and uniform spreading over the tissue section. Mounted sections are clear with an ideal refractive index suitable for high resolution oil immersion microscopy. VectaMount™ Mounting Medium preserves the color and intensity of preparations stained with enzyme substrates such as DAB, TMB, and BCIP/NBT, as well as our proprietary substrates Vector^® NovaRED™, Vector^® VIP, Vector^® SG, ImmPACT™ DAB, ImmPACT™ NovaRED™, ImmPACT™ VIP, ImmPACT™ SG, Vector^® Red, Vector^® Blue, and Vector^® Black. The crystal formation that frequently occurs with the alkaline phosphatase substrate BCIP/NBT using other permanent mounting media is essentially eliminated.

Tissue Staining with ImmPACT® DAB Substrate

ImmPACT™ DAB produces a dark brown reaction product that is crisper and is 3-4 times more sensitive than the original Vector DAB substrate kit. It can be used for both immunohistochemical and blotting applications and for manual and automated staining methods. The ImmPACT™ DAB working solution is stable for at least five days at room temperature and at least two weeks when stored at 4 °C. The ImmPACT™ DAB Substrate consists of 120 ml of diluent and a convenient concentrated stock solution of highly purified DAB and is stable for one year stored under refrigeration.

ImmPRESS® Polymer Detection Kit for IHC/ICC

The highly sensitive, one-step, ready-to-use, non-biotin, ImmPRESS™ Polymer Detection reagents from Vector Laboratories are the result of novel conjugation methods and micropolymer chemistry. Peroxidase micropolymers allow excellent resolution and crisp, strong staining of antigens, especially nuclear and membrane antigens. ImmPRESS™ reagents avoid the steric problems of other polymer-based reagents, thus providing superior results. These reagents are ideal for multiple antigen labeling because of the simplified one-step protocol.

Deparaffinization & Rehydration

Before paraffin sections can be stained in an immunohistochemical (IHC) procedure, your slides need to be deparaffinized and rehydrated. This tutorial will show you how to remove the wax (paraffin) with xylene or xylene substitute, then rehydrate your sections through a graded alcohol series.

Antigen Unmasking using Pressure Cooker Technique

The process of fixing and heating tissue sections in a formalin-fixation, paraffin-embedding procedure often buries epitopes so that they cannot be detected by your primary antibody. Vector Laboratories' Antigen Unmasking Solutions are highly effective at revealing antigens in formalin-fixed, paraffin embedded tissue sections when used in combination with a high temperature treatment procedure. These Antigen Unmasking Solutions are available in two formulations. The citrate-based solution (H-3300) is pH 6.0; the Tris-based solution (H-3301) is pH 9.0. Here we demonstrate pressure cooker technique for antigen retrieval.

Tissue Staining with VIP Peroxidase Substrate

Vector® VIP and ImmPACT™ VIP substrates both produce intense, violet reaction products, and can be used as alternatives to DAB or as a second color for multiple antigen labeling. Vector® VIP and ImmPACT™ VIP substrates also provide excellent color contrast in pigmented tissues such as melanoma. With the aid of imaging systems and software, the spectral profile of both substrates can be distinguished from other enzyme substrates in applications where antigens are co-localized. Sections stained with either substrate can be viewed by darkfield and electron microscopy. Vector^® VIP and ImmPACT™ VIP substrates can be used for both manual and automated staining methods. Stained sections should be dehydrated, cleared, and permanently mounted. The Vector^® VIP Substrate kit contains stock solutions in convenient dropper bottles. The sensitivity of Vector^® VIP substrate is equivalent to Vector^® DAB Substrate Kit (SK-4100).

Tissue Staining with SG Peroxidase Substrate

Vector^® SG and ImmPACT™ SG substrates produce blue-gray reaction products that can be used as a single label or as a second color for multiple antigen labeling. With the aid of imaging systems and software, the spectral profile of both substrates can be distinguished from other enzyme substrates in applications where antigens are co-localized. Sections stained with either substrate also can be viewed by darkfield or electron microscopy. Vector^® SG and ImmPACT™ SG substrates can be used for both manual and automated staining methods. Stained sections can be dehydrated, cleared, and permanently mounted. Vector^® SG Substrate Kit (SK-4700) contains stock solutions in convenient dropper bottles. The sensitivity of Vector^® SG subsrate is equivalent to AEC.

Tissue Staining with AEC Peroxidase Substrate Kit

AEC (3-amino-9-ethylcarbazole) substrate kit and ImmPACT™ AEC from Vector Laboratories both produce red reaction products that can be used as a single label or as a second color for multiple antigen labeling. AEC and ImmPACT™ AEC can be used for both manual and automated staining methods. Stained slides must be aqueously mounted because these reaction products are soluble in organic solvents. Sections stained with AEC are stable for at least 2 years when mounted in VectaMount™ AQ (H-5501).

Tissue Staining with NovaRED Peroxidase Substrate

Vector^® NovaRED™ and ImmPACT™ NovaRED™ substrates produce red reaction products. Unlike AEC, sections stained with either substrate should be dehydrated, cleared, and permanently mounted. Both substrates are useful as alternatives to DAB or as a second color for multiple antigen labeling. Vector^® NovaRED™ and ImmPACT™ NovaRED™ substrates also provide excellent color contrast in pigmented tissue such as melanoma. With the aid of imaging systems and software, the spectral profile of both substrates can be distinguished from other enzyme substrates in applications where antigens are co-localized. Sections stained with either substrate also can be viewed by darkfield or electron microscopy. Vector^® NovaRED™ and ImmPACT™ NovaRED™ substrates can be used for both manual and automated staining methods. Vector^® NovaRED™ Substrate Kit (SK-4800) contains stock solutions in convenient dropper bottles. The sensitivity of Vector^® NovaRED™ substrate is equivalent to Vector^® DAB substate (SK-4100), and 4 times greater than AEC.

VectaMount® AQ Mounting Medium for Aqueous Mounting of Sections

VectaMount™ AQ Mounting Medium (H-5501) from Vector Laboratories is a hardsetting mounting medium developed by Vector Laboratories for use with enzymatic substrates, such as AEC, ImmPACT™ AEC, and ImmPACT™ AMEC Red, whose reaction products are soluble in alcohol or other organic solvents. In applications where aqueous mounting is preferred, VectaMount™ AQ is suitable for use with other substrates such as DAB, Vector^® SG, BCIP/NBT, Vector^® Red, Vector^® Blue, ImmPACT™ DAB, and ImmPACT™ SG. VectaMount™ AQ is simple to use, requires no mixing, and preserves the color and clarity of the substrates. Stained sections mounted with VectaMount™ AQ can be stored in a slide box at room temperature for at least 2 years without fading.

Tissue Staining with DAB Peroxidase Substrate Kit

DAB (3, 3'-diaminobenzidine) produces a dark brown reaction product and can be used for both immunohistochemical and blotting applications. Vector DAB Substrate Kit produces a precipitate that is 2-4 times more sensitive than conventional DAB substrates (powders or tablets). DAB is effective as a single label or as a second color for multiple antigen labeling. Because of its heat-resistance, DAB can be used in IHC/ ISH double labeling applications. With the aid of imaging systems and software, the spectral profile of DAB can be distinguished from our other proprietary enzyme substrates in applications where antigens are co-localized. Sections stained with DAB can also be viewed by darkfield and electron microscopy. Slides developed with DAB can be dehydrated, cleared, and permanently mounted.

VECTASTAIN® Elite® ABC Kit for IHC/ICC

With more than 60,000 citations to its credit, the VECTASTAIN^® ABC kit from Vector Laboratories remains widely popular. Based on the versatile biotin/avidin interaction, the system is modular, and along with our selection of secondary antibodies, can accommodate a wide array of primary antibody and tissue species. Our ABC kits are economical and continue to be a staple product in any immunohistochemistry laboratory. The VECTASTAIN^® ABC systems are extremely sensitive due to the form and number of active enzyme molecules associated with the preformed Avidin/Biotinylated enzyme Complex. This complex takes advantage of two important properties of avidin: 1) an extraordinarily high affinity for biotin (over one million times higher than antibody for most antigens), and 2) four biotin-binding sites. These properties allow macromolecular complexes (ABC's) to be formed by mixing Avidin DH (Reagent A) with its paired biotinylated enzyme (Reagent B) prior to use. The ABC, once formed, remains stable for many hours after formation and can be used for several days after preparation.

Nucleic Acid Labeling with PHOTOPROBE® Biotin Kit

PHOTOPROBE^® Nucleic Acid Labeling System from Vector Laboratories is the method of choice for simple and rapid incorporation of biotin at multiple sites along the entire length of the nucleic acid. The nucleic acid labeling reaction does not destroy the original nucleic acid nor creates its copy. The integrity of the original sample is preserved. Single or double-stranded DNA, circular DNA, RNA, or PNA (peptide nucleic acid) can be labeled with the same reagents.

Troubleshooting background in IHC and IF applications

Although the causes of non-specific background staining in Immunohistochemistry (IHC) and Immunofluorescence (IF) applications may differ slightly, they can obscure the detection of your specific signal. Learn about the sources of background staining and how to troubleshoot unwanted background in IHC and IF.

Topics of discussion include:

Background inherent to the specimen
How the combination of specimen and detection reagents influences background
Autofluorescence

Designing Immunohistochemical Procedures

Part 1: Selecting Your IHC Reagents

Considerations for Designing Immunohistochemical Procedures

Part 2: Selecting Your IHC Reagents

Dot Blot Method

In applications involving several steps - from producing and labeling a probe to detecting the labeled probe - assessing labeling efficiency can be an essential part of assay design. The efficiency of labeling can be estimated by comparing the relative detection sensitivity of the labeled nucleic acid to a standardized sample in a side by side dot blot dilution series.

Immunohistochemistry using the VECTASTAIN® ABC Kit

Chi, V., Chandy, K. G. Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs. J. Vis. Exp. (8), e308, doi:10.3791/308 (2007).

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

Western Blotting using the VECTASTAIN® ABC Kit

Gallagher, S., Chakavarti, D. Immunoblot Analysis. J. Vis. Exp.(16), e759, doi:10.3791/759 (2008).

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon. The immunoblotting technique has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive (1 ng of antigen can be detected). This unit provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.