Introduction to Immunohistochemistry
Immunohistochemistry, or IHC, is the method for localizing specific antigens in tissue or cells using antibodies, enzyme conjugates and substrate-chromogens. Used in histology to assist with accurate tumor classification and diagnosis. IHC is an advanced staining technique, used to complement Hematoxylin & Eosin (H&E) and other staining techniques that typically show tissue morphology (structure). Compared to non-specific H&E stains, IHC is used to aid in the differential diagnosis and classification of cancers, and other diseases, including infections.
IHC of Ki-67 on FFPE Tonsil Tissue
Target Antigen
Antigens are proteins that are within or on the surface of a cell. an IHC diagnosis includes look for the presence or absence of particular antigens. Pathologist often use multiple antigens, or a “panel”, to help with a diagnosis.
Primary Antibody
There are two main types of antibodies used in immunohistochemistry, monoclonal and polyclonal. Polyclonal antibodies have an affinity with, and bind to, multiple epitopes (or parts) or the target antigen, and as such are more prone to cross-react to non-target antigens.
Monoclonal antibodies have an affinity to only one epitope and tend to produce cleaner, more specific staining but are less sensitive or intense.
Bio SB offers antibodies from both Mouse and Rabbit Monoclonal sources, and is proud to present one of the largest portfolios of rabbit monoclonal antibodies available in the molecular pathology industry. The Bio SB line of rabbit monoclonal antibodies offer better signal and less background than mouse monoclonal or rabbit polyclonal antibodies.
The Detection System
The detection system builds on the secondary. Modern chromogenic detection utilizes enzymes such as Horseradish Peroxidase (HRP) that are conjugated (joined) to an antibody. Multiple enzymes attached to the antibody are known as polymers, and they again produce more intense staining as there are more molecules for the chromogen to attach.
Bio SB offers a robust and diverse array of Biotin and Fab Micropolymer based detection systems for use in both research and clinical laboratories, with a variety of Biotin/Streptavidin & Fab Micropolymer detection chemistries.
The Chromogen
Finally, a substrate forms an insoluble colored precipitate that can be visualized under a microscope. commonly used chromogens are DAB Brown or AEC Red.
DAB Brown is most common, as it provides strong and permanent stains. AEC Red (or HRP Green) is used mainly for skin sections where the the DAB Brown may get confused with residual melanin in the tissue.
Multiple different colors are sometimes used in the same tissue section to allow the pathologist to visualize two antigens in the one slide, in a process known as Multiplex IHC Staining.
The Counterstain
The background color of the tissue is deposited by the a hematoxylin counter-stain, applied after the chromogen. The counter-stain provides a contrast to the chromogen and also helps the pathologist visualize the underlying tissue structure. Standard hematoxalin provides a blue background, we also offer a Pink Hematoxylin that provides a pink background, ideal for multiplex purposes or with when using Skin tissue with residual melanin.
Simple Tips to Solve Common IHC Problems In Your Lab.
Poor Tissue Adhesion
Poor Tissue adhesion leads to detachment, air bubbles, wrinkling and tissue damage. Stable and strong adhesion of a tissue specimen to the microscope slide surface is important for achieving successful and consistent sample preparation and IHC staining.
Use our Bio SB Hydrophilic Plus Slides with over 3x as many charges to prevent tissue detachment and promote reagent dispersal over the entire working area of the slide. This ensures consistent and uniform results in Immunohistochemistry and a clearer picture of the tissue morphology.
Residual Paraffin
Microscopic paraffin residue can remain, even after HEIR and deparaffination steps are taken. This microparaffin residue can effect the specificity and sensitivity of the IHC signals.
Using our Bio SB ChromoProtector before mounting slides has been shown to completely eliminate paraffin or micro-paraffin residue.
Bio SB ChromoProtector also protects substrate-chromogens that are soluble or fade with solvents prior to mounting with solvent mounting media and thus allows for a solvent-free environment when conducting IHC procedures.
Non-Specific Staining
Low affinity mouse monoclonal and rabbit polyclonal antibodies can sometimes cause to non-specific signals and background staining, which leads to an unclear diagnosis.
Bio SB’s Rabbit Monoclonal antibodies can have 10-100 times higher affinity than some mouse monoclonals for clear IHC results. Bio SB is proud to present over 220 Rabbit Monoclonal antibodies, one of the largest portfolios of RMab’s available in the molecular pathology industry.
Cyclin D1, MMAb
Cyclin D1, RMAb
Weak Nuclear Detection
Weak nuclear signals and excessive background staining can be caused by low quality detection system chemistry.
Use our Bio SB Fab Mouse and Rabbit Polydetector Plus Micropolymer Detection System with a much smaller micro-polymer molecule compared to our competitors. You can achieve reliable, highly specific and sensitive nuclear staining, even on tough targets!
The PolyDetector Plus IHC Detection System incorporates an Immunoglobulin link and a Fab Micropolymer label. This multiple component Fab Micropolymer delivers a highly sensitive and specific signal even in a shorter time-frame than comparable polymeric detection systems.
Validation Needs
Finding simple, reliable means of validation is becoming increasingly important for In-vitro diagnostic and research labs.
Bio SB manufactures our own Tissue and Cell Line Microarrays in a variety of formats for all of your validation needs. Including normal tissue, various cancer tissues and cell lines, as well as hard-to-find Infectious Disease Cell Line Microarrays.
