Histology Kits: Avidin and Biotin Team Up to Tackle the Tissue

Date: May 25, 1998 Author: Shane Beck Charts For those researchers who wish to delve into cells and tissues, there are several immunohistochemical and immunocytochemical detection methods to choose from. From the antibody-enzyme conjugation and peroxidase antiperoxidase (PAP) methods pioneered in the late 1960s came the development of avidin-biotin affinity techniques. Taking advantage of avidin's high affinity for biotin, scientists were, and still are able to look at tissues in new ways. Ear

By | May 25, 1998

Date: May 25, 1998
Author: Shane Beck
For those researchers who wish to delve into cells and tissues, there are several immunohistochemical and immunocytochemical detection methods to choose from. From the antibody-enzyme conjugation and peroxidase antiperoxidase (PAP) methods pioneered in the late 1960s came the development of avidin-biotin affinity techniques. Taking advantage of avidin's high affinity for biotin, scientists were, and still are able to look at tissues in new ways. Early research in avidin-biotin cytochemistry by H. Heitzman and F. Richards ( Proceedings of the National Academy of Sciences U.S., 71:3537-41, 1974) and Edward A. Bayer, Meir Wilchek, and colleagues (E.A. Bayer et al., Journal of Histochemistry and Cytochemistry, 24:933-9, 1976; E.A. Bayer and M. Wilchek, Methods in Biochemical Analysis, 26:1-45, 1980) led to the development of immunohistochemical techniques still in use today.

In the 1920s, scientists discovered the protein avidin in egg white. Avidin is a tetramer with an extremely high affinity for biotin, possessing a dissociation constant of approximately 10-15 M. Biotin is a coenzyme of decarboxylase present in a variety of tissues, including liver, intestine, pancreas, and kidney. Avidin possesses four binding sites for biotin, and since biotin can be coupled with a wide variety of reporter groups, including enzymes, fluorescent dyes, or radioactive isotopes, the detection capabilities of this system seem limitless. Streptavidin, an avidin analog produced by the bacterium Streptomyces avidinii, also exhibits a strong affinity for biotin and is commonly used as a reporter in place of avidin. Since streptavidin has a near neutral isoelectric point and has no carbohydrate residues, it exhibits less nonspecific binding than avidin. While not included in any of the kits profiled in this article, NeutrAvidin™, a biotin-binding protein provided by Pierce has a more neutral isoelectric point than streptavidin.

Immunohistochemical stain of an epithelial tumor using an anti-cytokeratin antibody. Red deposition of the chromogenic substrate 3-amino, 9-ethylcarbazole identifies the cytokeratin-containing neoplasm (Biomeda).
Avidin and biotin can be used in several manners for immunohistochemical detection. The first detection method is the avidin-biotin complex (ABC) technique. ABC takes advantage of avidin's four binding sites for biotin conjugates. By optimizing the ratio of avidin and biotinylated enzyme, macromolecular avidin-biotin complexes can be formed. A variation of this method was described by Su-Ming Hsu in 1981 (S.M. Hsu et al., American Journal of Clinical Pathology , 75:734, 1981). Another method, dubbed the labeled avidin-biotin (LAB) technique, uses an avidin conjugate, such as avidin-peroxidase, to visualize a biotin-labeled substance. Lastly, in the bridged avidin-biotin (BRAB) method, an avidin-biotin sandwich is made when avidin is used to link two or more biotin-conjugated molecules. This method is theoretically more sensitive than the LAB method due to increased amplification by the biotin-avidin-biotin interaction. For more information on avidin-biotin chemistry, two pieces by Wilchek and Bayer detail various aspects of this technology (M. Wilchek, E. A. Bayer, Analytical Biochemistry, 171:1-32, 1988; M. Wilchek, E.A. Bayer, Methods in Enzymology, 184:3-363, 1990).

The following synopsis of products for histology focuses on those that utilize avidin-biotin technology, as well as some unique kits that use biotinyl tyramide for signal amplification and detection. Most kits employ horseradish peroxidase (HRP) or alkaline phosphatase (AP) as the tracers, but some kits now available use ß-galactosidase, glucose oxidase, and phycoerythrin. Aside from differences in methodologies among these products, there is some variability in kit components, as well. Some kits provide just about everything you would need, including necessary buffers, mounting media, and chromogenic substrates for detecting the labeled enzymes. Common chromogen-substrates for detecting AP include 5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt (BCIP), nitro-blue tetrazolium chloride (NBT) and Fast Red. BCIP and NBT usually are used together to yield a dark purple precipitate, while Fast Red, as indicated by its name, produces a bright red precipitate. Those substrates employed for visualizing HRP include 3-amino-9-ethyl carbazole (AEC) and 3,3'-diamino benzidine tetrahydrochloride (DAB). AEC will produce a reddish end product that is soluble in alcohol, while DAB will generate a brown end product.

Also on the market are a few specialized kits, such as those for probing estrogen receptor and progesterone receptor. Now that you know the ABCs of immunohistochemistry, here are some products that are designed to take some of the pain out of staining.

BioGenex markets a variety of biotin-streptavidin-based kits for immunodetection. These kits include individual kits matching the animal species of the primary antibody and MultiLink® kits that contain a universal secondary antibody that can detect most monoclonal and polyclonal primary antibodies. Kits are also available in a variety of configurations, including Ready-to-Use, Large Volume, and Concentrated; BioGenex also sells kits prepackaged for use with the BioGenex OptiMax® Plus Automated Cell Stainer.

Super Sensitive Ready-to-Use Detection Systems
The Super Sensitive Ready-to-Use Detection Systems utilize BioGenex's proprietary amplified biotin-streptavidin technology for high sensitivity and low background on paraffin sections. Individual kits are available with either species-specific or MultiLink secondary antibodies, suitable for mouse, rabbit, guinea pig, and rat primary antibodies. In addition, all 50-slide kits are available with alkaline phosphatase (Fast Red chromogen) or peroxidase (DAB or AEC chromogen) as the enzyme label.

High Performance Ready-to-Use Detection Systems
The High Performance Ready-to-Use Detection Systems are designed for immunostaining with standard antibody dilutions with low background using BioGenex's streptavidin-based technology. While not as sensitive as the Super Sensitive Systems, these kits still provide a reliable detection system for routine immunostaining. Kits are available with either species-specific or MultiLink antibodies, and alkaline phosphatase (Fast Red chromogen) or peroxidase (AEC chromogen) as the enzyme label.

Super Sensitive Concentrated Detection Systems
For maximum economy and flexibility, BioGenex offers the Super Sensitive Concentrated Detection Systems, which come with concentrated link and label reagents. The concentrated reagents can be diluted to produce a working solution of 100 ml (for Super Sensitive antibodies) to 325 ml (for use with High Performance antibodies). This kit is ideal for weakly expressed antigens, such as estrogen receptor and progesterone receptor. All kits come with alkaline phosphatase or peroxidase enzyme label.

Super Sensitive Animal Detection Kit
Lastly, BioGenex now offers a selection of biotin-streptavidin-based detection systems optimized for immunostaining animal specimens. The Super Sensitive Animal Detection Kit reagents have been treated for solid-phase adsorption to help eliminate interfering cross-reactivities. Kits are compatible with rat, rabbit, and mouse tissues.

BioStain Super ABC Kits
The BioStain Super ABC Kits can be used for the detection of biotin-labeled DNA hybridization probes, hybridoma screening, solid phase enzyme immunoassays, DNA and protein detections in transfer blots, immunocytochemistry, and other immunoassay applications. Utilizing ABC technology, these kits are available with alkaline phosphatase, phycoerythrin, phycocyanin, or peroxidase.

Immunoperoxidase staining of formalin-fixed, paraffin-embedded normal human tonsil at high magnification showing membrane localization of tyrosine phosphorylated proteins (Santa Cruz Biotechnologies).
Multi-species AutoProbe™ Immunocytochemistry Systems
The AutoProbe Detection Systems combine the technologies of horseradish peroxidase tracing with the streptavidin-biotin complex (s-ABC) and Biomeda's wide-spectrum multi-animal recognition system. These kits simultaneously recognize primary antibodies raised in nine different animal species (monoclonal mouse and rat, polyclonal guinea pig, rabbit, goat, sheep, cow, donkey, and horse). AEC is supplied as the chromogen in the AutoProbe III Kits, while the AutoProbe II Kits utilize DAB.

Multi-species UltraProbe™ Immunocytochemistry Systems
Combining alkaline phosphatase with ABC technology, the UltraProbe Detection Systems also take advantage of Biomedaís wide-spectrum multi-animal recognition system, which reduces the potential for human error in procedural steps. Fast Red is supplied as the chromogen. Biomeda also supplies UltraProbe Basic Kits for detection of biotinylated DNA probes for in situ hybridization and the detection of biotin-labeled proteins in transfer blots.

LAB/Probe Kit
The LAB/Probe Kit is a labeled streptavidin-biotin HRP kit with mouse and rabbit primary antibody recognition. The rapid room temperature protocol specifies a 10-minute primary antibody incubation, a 10-minute secondary antibody incubation, and a 10-minute streptavidin-HRP incubation. The kit is available with either AEC or DAB as the chromogen.

HistoScan/LymphoScan Immunohistochemical Detection Systems
Based on ABC technology and using peroxidase as a tracer, the HistoScan/LymphoScan Systems are available for the detection of a broad spectrum of human antigens, hormones, and infectious agents in formalin-fixed, paraffin-embedded tissue sections. These kits also work with frozen sections and cytological specimens, such as smears and cytospins. Biomeda also supplies Specific Alkaline Phosphatase Kits, similar to the HistoScan/ LymphoScan Systems but the enzyme used is AP. These kits are recommended for tissues containing high levels of endogenous peroxidase. Also available are the Specific Immunophycoerythrin Kits. These kits permit clear discrimination between the yellow-orange signal and the greenish autofluorescence background.

Universal HistoScan Kits
Using a direct streptavidin-enzyme conjugate approach, Biomeda provides three separate Universal HistoScan Kits. These kits differ from the other HistoScan Kits in that no primary antibody is included in the Universal Kits. Available are the Universal Immunoperoxidase Staining Kits, Universal Phycoerythrin Staining Kits, and Universal Immunoalkaline Phosphatase Staining Kits.

DAKO Corporation
DAKO Catalyzed Signal Amplification System
DAKO's Catalyzed Signal Amplification System (CSA) is based on the peroxidase catalyzed deposition of biotinyl tyramide. When oxidized by peroxidase, biotinyl tyramide is converted into an intermediate that is highly reactive with electron-rich aromatic amino acids in proteinaceous specimens. Deposition of biotinyl tyramide is followed by HRP-conjugated streptavidin binding that results in high amplification of biotin signals at the binding site of biotinylated antibodies. By using CSA, reproducible detection of low level antigen expression can be achieved. CSA enhances weak antibody reactions, enables reactions of antibodies previously unreactive on formalin-fixed paraffin-embedded specimens, and works with monoclonal and polyclonal antibodies.

Diagnostic BioSystems
Universal Immunostaining Kits
Based on streptavidin biotin technology, the Universal Immunostaining Kits are available with peroxidase or alkaline phosphatase as the tracer enzyme. Kits are available in two sizes; small volume kits allow 50 to 100 tests while large volume kits have sufficient reagents for 500 to 1,000 tests. Small volume kits offer a choice of DAB or AEC as the chromogen, whereas the large volume kits do not contain a chromogen. Alkaline phosphatase kits come with Fast Red as the chromogen.

Diagnostic BioSystems has developed a kit for immunocytochemical localization of estrogen receptor (ER) and progesterone receptor (PR) in formalin-fixed paraffin-embedded tissue sections. Presence of ER and PR in breast tissue is positively correlated with a prolonged disease-free interval. Thus, receptor-rich tumors are more likely to respond to endocrine therapy than to chemotherapy.

The streptavidin-biotin-based ER/PR Kit comes with ready-to-use reagents, uses DAB as a chromogen, and has a high signal-to-noise ratio. Treated tissues can be dehydrated for permanent record, and the total time for immunostaining is less than three hours. The kit comes with enough reagents for 50 tests for each antibody.

Harlan Bioproducts for Science
SEEKit Biotin Streptavidin Systems

Harlan's SEEKit Biotin Streptavidin Systems are for use with cryostat or formalin-fixed, paraffin-embedded sections. The sensitivity of the kits allow the detection of proteins and nucleic acids blotted from gels to nitrocellulose membranes. The Universal SEEKit System includes streptavidin and biotinylated HRP. The Anti-Immunoglobulin SEEKit Systems are designed for detection and/or quantification of mouse, rat, or rabbit primary antibodies in tissue sections, Western blots, or ELISAs. The Anti-Immunoglobulin SEEKits come with biotinylated primary antibody, streptavidin, and biotinylated HRP. Harlan offers sample sizes of all SEEKit Systems.

ICN Biomedicals
Immu-Mark™ Universal Kits
For identifying tissue antigens using the streptavidin-biotin immunostaining method, ICN offers the Immu-Mark Universal Kit basic detection systems. Kits are available with alkaline phosphatase or horseradish peroxidase, and are for use with mouse, rabbit, and goat primary antibodies. Each kit contains ready-to-use biotinylated link antibody with blocking reagent incorporated and enzyme-labeled streptavidin for use with the antibody of the user's choice. The Universal Combination Species kit is suitable for use with antibodies from all three animal species.

UltraTech Streptavidin-Biotin System
Available from Immunotech (a division of Coulter Corporation) are the UltraTech-HRP and UltraTech-AP Systems, based on streptavidin-biotin technology. These kits are available in a 70-test format and 500 to 600-test configuration. Included with the large volume kits are streptavidin-peroxidase or streptavidin-alkaline phosphatase, biotinylated multivalent secondary antibody, and blocking agent. A three-step procedure specifies a 10-minute secondary antibody incubation, a 10-minute streptavidin-enzyme complex incubation, and incubation with chromogen. Available separately are UltraTech-AEC, UltraTech-DAB, and UltraTech-Fast Red 70-test kits. These kits contain either streptavidin-peroxidase or streptavidin-alkaline phosphatase, biotinylated multivalent secondary antibody, blocking agent, hydrogen peroxide for peroxidase kits, associated chromogen, and substrate buffer.

Immunoperoxidase staining of formalin-fixed, paraffin-embedded human tongue tissue stained with monoclonal anti-alpha-sarcomeric actin (Sigma).
Kirkegaard & Perry Laboratories
HistoMark® Universal Streptavidin Kits
Kirkegaard & Perry Laboratories offers three streptavidin-biotin-based kits for the rapid and precise localization of cell surface and intracellular antigens in frozen or paraffin-embedded tissue, cytospins, and touch preparations. For use with antibodies produced in mouse, rabbit, and rat, KPL provides the Streptavidin-HRP Systems, the Streptavidin-AP Systems, and Streptavidin-ß-Galactosidase Systems. The Streptavidin-ß-Galactosidase Systems are designed for applications where endogenous peroxidase or phosphatase are potential trouble areas. The HistoMark universal reagents are in a prediluted, ready-to-use format in dropper bottles. Included with the kits, the biotinylated link antibody has a thirteen-atom spacer arm between the biotin ring and the antibody to minimize steric interactions with streptavidin and maximize the streptavidin-biotin binding reaction. Also included with the kits are streptavidin-enzyme reagent and serum block. KPL provides recommended HistoMark substrates separately.

NEN Life Science Products
Renaissance® TSA™ Systems
The Renaissance TSA Systems take advantage of NEN's patented catalyzed reporter deposition technology. TSA Systems utilize HRP to catalyze the deposition of biotinyl or fluorescent tyramide onto tissue sections or cell surfaces that were previously blocked with protein. This reaction occurs in less than 10 minutes and causes a covalent fluorochrome (TSA-Direct) or biotin deposition (TSA-Indirect) directly at the enzyme site, resulting in profound signal amplification. Company literature claims signal amplification of up to 100 times greater than standard chromogenic or fluorescent detection. As long as HRP is or can be an integral part of the detection protocol, TSA can be used. Streptavidin-HRP is included in the TSA kits for use with biotin-labeled antibodies or probes.

TSA Systems are available for fluorescence and chromogenic in situ hybridization and immunohistochemistry. Each kit includes streptavidin-HRP conjugate, NEN blocking reagent, amplification diluent, and biotnyl- or fluorescent-tyramide. TSA-Indirect comes in 50 and 200 slide kits, and TSA-Direct kits are for 50 or 100 slides.

ImmunoPure® ABC Staining Kits
Pierce provides ImmunoPure ABC Staining Kits with HRP and AP which are available with or without a biotinylated secondary antibody. The ImmunoPure Standard Kit includes only avidin and biotinylated enzyme, and is useful if an ABC kit is not available for your specific species of primary antibody or if the specificity of the antibody in the kit is not appropriate for the application. In instances such as these, the biotinylated antibody for your specific application can be purchased separately and used with the standard kit. The Ultra-Sensitive ABC Peroxidase Staining Kits are more sensitive than the ABC Peroxidase Staining Kits and exhibit no increase in background staining, ideal for localizing antigens present in small quantities. Kits with a secondary antibody are selected by the species of primary antibody to be used.

Research Diagnostics Inc.
POLY-HRP-Detection Technology
Though not an avidin-biotin-based kit, RDI's POLY-HRP-Streptavidin is composed of a proprietary polymer of HRP conjugated to streptavidin in various polymerization ranges. The POLY-HRP-Streptavidin product line can be used with most biotin labeled products, including probes and primary and secondary antibodies. These products are suited for immunohistochemical/ cytochemical protocols, and they are especially effective in the detection of minor surface membrane antigens. POLY-HRP conjugates increase the number of HRP molecules available to react with substrate by greater than five times, increasing sensitivity.

Santa Cruz Biotechnology
ABC Staining Systems
New to Santa Cruz Biotechnology's product line, the ABC Staining Systems (mouse, rabbit, rat, and goat) include normal blocking serum, biotinylated secondary antibody, avidin-biotinylated-HRP, peroxidase substrate, DAB chromogen, and DAB dilution buffer. Tissue sections are first incubated with primary antibody, then biotinylated secondary antibody is added. Lastly, preformed ABC reagent is added and the target protein is visualized by incubation in the peroxidase substrate.

ImmunoCruz™ Staining Systems
ImmunoCruz Staining Systems include negative control IgG, peroxidase blocking solution, normal blocking serum, biotinylated secondary antibody, streptavidin-HRP reagent, peroxidase substrate, DAB chromogen, and DAB dilution buffer. All reagents are prediluted and ready to use for immunohistochemical tissue staining. As an option, kits can be supplied with prediluted primary antibody.

Scytek Laboratories
SensiTek Staining System
Scytek's SensiTek Staining System is for immunohistochemical staining and comes in a simple, ready-to-use format. The SensiTek kits are available with either HRP or AP conjugated to streptavidin, and the chromogens are provided in liquid form to increase user safety whenever possible. These kits require a 20-minute incubation time for both the link antibody and the enzyme label. The Anti-Polyvalent is suited for use with mouse, rat, rabbit, or guinea pig antibodies. The 70-slide kits come with Peroxide Block, Super Block, Link Antibody, Enzyme Label, and DAB, AEC, or Fast-Red chromogen; the 500-slide kits come with Super Block, Link Antibody, and Enzyme Label.

ExtrAvidin® Peroxidase Staining Kits
ExtrAvidin is a uniquely modified avidin reagent, allowing high specific activity and sensitivity with low background staining of streptavidin. Kits are comprised of universal reagents for use with primary antibodies in immunohistology, ELISA, and immunoblotting. The biotinylated, affinity isolated antibody supplied with the kit is conjugated using a spacer to allow greater accessibility for the ExtrAvidin-Peroxidase.

Each kit consists of biotinylated secondary antibody (goat, mouse, rabbit, or rat), ExtrAvidin-Peroxidase, and directions for use. The Goat ExtrAvidin Peroxidase Staining Kit may also be used with sheep primary antibodies, and the Mouse ExtrAvidin Peroxidase Staining Kit reacts with mouse IgG and IgM. The kit for immunohistology comes with reagents sufficient for 500 tests.

ExtrAvidin Alkaline Phosphatase Staining Kits
The ExtrAvidin Alkaline Phosphatase Staining Kit consists of biotinylated secondary antibody (goat, mouse, or rabbit), ExtrAvidin-Alkaline Phosphatase and directions for use with each application. Like its peroxidase partner, the biotinylated antibody supplied in this kit is conjugated using a spacer to allow greater accessibility for the ExtrAvidin-Alkaline Phosphatase. The Goat ExtrAvidin Peroxidase Staining Kit may also be used with sheep primary antibodies, and the Mouse ExtrAvidin Peroxidase Staining Kit reacts with mouse IgG and IgM. The kit for immunohistology comes with reagents sufficient for 500 tests.

Immunoperoxidase staining of formalin-fixed, paraffin-embedded human tongue tissue stained with monoclonal anti-alpha-smooth muscle actin using Mouse ExtrAvidin Staining Kit (Sigma).
Rapid Staining Kits
Specifically for use with high affinity antibodies, Sigma's Rapid Staining Kits utilize the avidin/biotin detection system with ExtrAvidin-Peroxidase. These kits are designed for identification of antigens in formalin-fixed, paraffin-embedded tissue sections. Labeling can be completed in less than 20 minutes.

Each kit contains reagents for approximately 50 slides and consists of ready-to-use biotinylated secondary antibody, ExtrAvidin-Peroxidase reagent, substrate buffer, hydrogen peroxide, AEC chromogen, and instructions.

Immunohistology Kits
Sigma's Immunohistology Kits are based on the avidin/ biotin system using ExtrAvidin-Peroxidase. The kits are designed for identification of human tissue components in formalin-fixed, paraffin-embedded sections.

Each kit comes with primary antibody, biotinylated secondary antibody, ExtrAvidin-Peroxidase reagent, substrate buffer, hydrogen peroxide, and AEC chromogen. Negative control serum and blocking serum are not included.

Signet Laboratories
Signet USA™ Ultra Streptavidin Detection Systems
Formulated with either HRP or AP, Signet USA Ultra Streptavidin Systems incorporate Signet's Ultra Streptavidin. The USA Ultra Detection Systems are designed to individually detect rabbit and mouse primary antibodies; there is also a multispecies format that allows for the detection of both rabbit and mouse primary antibodies using the same system. The 50-test kits come with blocking reagents, biotinylated secondary antibody, Ultra Streptavidin-HRP or Ultra Strep- tavidin-AP, AEC or DAB chromogen, and substrate buffer. The larger volume 150- and 500-test kits include blocking reagent, biotinylated secondary antibody, and Ultra Streptavidin-HRP or-AP.

Signet Genesis™ Starter System
Signet has developed the Signet Genesis Starter System to help beginning immunohistochemists gain familiarity with immunohistochemical techniques. Included with Genesis Starter Systems is the Signet USA Ultra Streptavidin Multi-Species Detection System (users can choose among HRP/AEC, HRP/DAB, and AP/Fast Red formats). Also included are Signet Level 2 ready-to-use primary antibodies, PBS wash buffer, ready-to-use Target Unmasking Fluid, Mayer's hematoxylin counterstain, mounting medium, and two staining trays.

Vector Laboratories
The VECTASTAIN ABC Kits from Vector Laboratories contain Avidin DH and biotinylated enzyme. Vector provides two peroxidase-based versions of the VECTASTAIN ABC kit. The VECTASTAIN Elite ABC kit provides five to ten times more sensitivity than the standard VECTASTAIN ABC kit. When the avidin and biotinylated peroxidase in the VECTASTAIN Elite ABC kit conjugate, a smaller, uniform, and highly active complex is formed, allowing a greater number of enzyme complexes to diffuse through a tissue section and bind to a biotinylated target. This kit is ideally suited for staining target substances in neural tissues such as brain.

The VECTASTAIN ABC-AP system uses alkaline phosphatase and is ideal for morphological examination of stained cells. If endogenous alkaline phosphatase is a problem, levamisole can be added to the substrate solutions during staining.

Though most sections do not express endogenous peroxidase or alkaline phosphatase activities, some instances may occur in which these endogenous enzymes interfere with specific staining. Since endogenous glucose oxidase activity is not present in mammalian tissues, Vector Laboratories has developed the VECTASTAIN ABC-GO kit. This kit, however, does not provide the sensitivity of the other VECTASTAIN ABC systems.

Lastly, Vector provides the VECTA STAIN Universal Quick Kit for rapid immunohistochemical staining of frozen or paraffin-embedded tissue sections. Using this system, staining of tissue sections following incubation with primary antibody can be accomplished in as little as 15 minutes. The Quick kit comes with streptavidin-peroxidase preformed complex, normal blocking serum, and a universal biotinylated antibody that recognizes mouse, rabbit, and goat antibodies.

Histostain™-Plus and HistoST5050™ Kits
Utilizing Zymed's second generation LAB-SA (labeled streptavidin-biotin) technology, the Histostain-Plus and HistoST5050 kits are designed for general all-around immunohistostaining applications. The Histostain-Plus kits are for both manual and automated immunohistochemistry techniques, while the HistoST5050 kits are designed and optimized specifically for the LEICA ST 5050 automated immunostainer. Kits come with streptavidin-HRP conjugate, biotinylated secondary antibody, blocking solution, and Zymed's exclusive Single Solution Chromogen system.

Histostain™-SP and Histostain™-SAP Kits
Also employing Zymed's second generation LAB-SA technology, the Histostain-SP and Histostain-SAP kits use streptavidin-peroxidase and streptavidin-alkaline phosphatase, respectively. Kits come with streptavidin-HRP (Histostain-SP kits) or streptavidin-AP (Histostain-SAP kits), biotinylated secondary antibody, blocking solution, substrate buffer, chromogen solution, hydrogen peroxide, hematoxylin solution, and mounting solution. DAB or AEC chromogens are used with the Histostain-SP kits, while the Histostain-SAP kits utilize AP-Red or BCIP/NBT.

Histostain™-DS Kit
Designed for immunohistochemical detection of two antigens in the same human cell or tissue sample, the Histostain-DS Kit utilizes double LAB- SA technology. The antigens can be identified using two different primary antibodies from the same animal species, or from different species. Streptavidin-alkaline phosphatase is used with BCIP/NBT (purple-black) to produce one stain, while streptavidin-horseradish peroxidase is used with AEC (reddish-brown) to produce a second stain. According to company literature, the two stains are easy to differentiate. The kit comes with blocking solution, streptavidin-AP and streptavidin-HRP, biotinylated secondary antibody, blocking solution, substrate buffers, NBT solution, BCIP solution, AEC chromogen, hydrogen peroxide, double staining enhancer, and mounting solution.

Histostain™-ER/PR Kits
These kits come with all necessary reagents for the detection of estrogen and progesterone receptors in formalin-fixed, paraffin-embedded tissues. Zymed provides a kit for the detection of estrogen receptor, a kit for the detection of progesterone receptor, and a kit that can detect both receptors. Included with the kits are streptavid in-peroxidase conjugate, biotinylated secondary antibody, mouse anti-ER or anti-PR antibody (or both), concentrated substrate buffer, concentrated DAB chromogen, hydrogen peroxide, hematoxylin solution, and HistomountTM. Utilizing the LAB-SA method, tissue is probed with ER or PR antibody, followed by incubation with a biotinylated secondary antibody. Streptavidin-enzyme conjugate is then added, and visualization occurs using the chromogen/substrate system.

BrdU and PCNA Kits
Bromodeoxyuridine (BrdU) is incorporated into proliferating cells during S-phase, similar to radioactive thymidine, and is a useful tool for studies of normal and neoplastic tissues. PCNA is similarly used to study proliferating cells. Zymed's BrdU and PCNA staining systems use a biotinylated monoclonal anti-BrdU or anti-PCNA antibody, eliminating the need for a species-specific secondary antibody. This allows staining to be performed in rabbits, rats, and mice without cross-reactivity problems. Streptavidin-peroxidase is used as the signal generator in these systems.

For immunohistochemical detection of tartrate-resistant acid phosphatase (TRAcP) in human tissues or cell preparations, the TRAcP Kit utilizes streptavidin-peroxidase and LAB-SA detection technology. TRAcP has been implicated in hairy cell leukemia and other maladies. The kit can be used on frozen tissues, paraffin-embedded sections, bone marrow preparations, and cell smears.

HistoMouse™ Kits
The HistoMouse-SP Kit is designed to detect mouse primary antibody on mouse tissue samples without causing background due to endogenous Ig. Zymed's exclusive BEATTM technology is used to prevent the secondary antibody from reacting with endogenous Ig. The LAB-SA detection method is used to amplify the signal, and AEC is used to create a strong red positive stain.


The author can be reached at sbeck@the-scientist.com

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