Buffers and Diluents

Buffers and diluents are available for Immunohistochemistry, in situ Hybridization Special Stains and most other applications.

        • General buffers, such as PBS (PH 7.6) and TBS (PH 7.6, 0.1M) can be used for washing/rinsing of slides.
        • Super SensitiveTM Wash Buffer is a phosphate buffered saline (PH 7.4) with surfactant and is used to ensure optimal staining with even spreading of antibodies and other reagents to avoid inconsistent results.
        • Common Antibody Diluent and Enhanced Antibody Diluent have been developed for use with all antibodies in immunohistochemistry and have been specifically optimized for use with BioGenex antibodies and reagent products. These diluents enable enhancement of signal-to-noise ratio of staining when used optimally and also help in maintaining the antibody specificity and stability. The Enhanced Antibody Diluent, in addition to all the above features, contains chemical compounds to enhance antigen-antibody interaction and affinity. In order to achieve desired staining pattern and intensity, the titers of antibodies or concentrations of reagents may need to be optimized. These diluents are also for diluting concentrated Alkaline Phsophatase (AP) labels but are not suitable for diluting Horseradish Peroxidase (HRP) labeles because they contain Sodium Azide (0.09%).
        • Streptavidin Peroxidase Diluent, was developed especially for diluting concentrated HRP labeles and does not contain Sodium Azide.
        • Link Diluent, was developed for diluting concentrated Link (Biotinylated Anti-Immunoglobulins) antibodies.

 

Blocking Reagents

                    • Peroxide Block: Endogenous peroxidase is most commonly encountered in red blood cells, kidney, and liver tissue. Peroxide Block should be used prior to application of primary antibody when Horseradish Peroxidase (HRP) is used as the labeling enzyme, and if it is necessary to block endogenous peroxidase activity in the tissue being stained.
                    • Power BlockTM: This is a blocking reagent for reducing nonspecific background in immunoassays. A truly universal block, it is suitable for use in immunohistochemistry, immunocytochemistry, ELISA methods, and immunogold techniques. The Power Block reagent contains buffer, casein and preservative and also works well as an antibody diluent and washing medium.
                    • Protein Block: This can be used to reduce background staining due to non-specific binding of the primary or secondary antibodies to the tissue. Protein Block should be applied immediately prior to the primary antibody.

 

Counterstains and Mounting Media

BioGenex offers the following counterstains for use in Immunohistochemistry, in situ Hybridization and other applications with either manual or automated staining systems.

                        • Mayer’s hematoxylin is a blue stain that does not contain alcohol and therefore is compatible with both alcohol soluble non-permanent chromogens (AEC, Fast Red and New Fuchsin) and alcohol-insoluble chromogens (DAB and Elegance Red). It is alcohol and xylene insoluble and therefore compatible with most clearing agents and mounting media.
                        • DAPI (4',6-diamidino-2-phenylindole) is a fluorescent blue stain used as a nuclear counterstain in Fluorescent In Situ Hybridization (FISH) and Immunofluorescence (IF) applications. DAPI strongly binds A-T rich regions in DNA and can be used to stain nuclei of both live and fixed cells. DAPI has an absorption maximum at a wavelength of 358 nm and its emission maximum is at 461 nm.

 

Mounting of all stained biological specimens is an essential step before their microscopic evaluation. Mounting also enables the slides to be archived for long periods of time. The mounting medium may be used to attach a coverslip or may itself serve as a coverslip substitute. The choice of mounting medium depends on whether long-term or short-term preservation is desired, and whether the mounting procedure is chemically compatible with the chromogen and the counterstain.

                      • SuperMount® Permanent Mounting Medium is a polymer based aqueous mounting media that does not require the use of a coverslip. This innovative, patented mounting medium (BioGenex's U.S. Patent No. 5,492,837) is designed to preserve biological specimens for long-term storage. SuperMount medium is compatible with most aqueous and organic-soluble dyes and chromogens including AEC, DAB, Elegance Red, Fast Red, New Fuchsin, BCIP/NBT, Rhodamine, Fluorescein, Texas Red, Phycoerythrin, Phycocyanin, and Fat Stain (Oil Red O). The refractive index of SuperMount yields greater transparency and clarity of specimens to be examined under the microscope. SuperMount can be used for the mounting of all biological specimens, including stained tissue sections, Cytospin preparations, and blood smears.
                      • Aqueous Mounting Medium is glycerol-based mounting medium that require the use of a coverslip. It is intended for short-term specimen storage and is compatible with most chromogens and counterstains.
                      • XMountTM Mounting Medium is a permanent mounting medium that has been optimized for use with BioGenex XMount instrument for all BioGenex detection systems for immunohistochemistry (IHC), In Situ Hybridization (ISH) and special stains. XMount is intended for use with alcohol and xylene insoluble chromogens, such as DAB (for peroxidase systems) and Elegance Red (for alkaline phosphatase systems). XMount dries clear with an ideal refractive index similar to high quality glass and tissue elements. Mounted slides can be viewed with high magnification oil immersion lenses. Also, when mounting preparations stained with the BCIP/NBT substrate, crystal formation that may occur when using other media is minimized.

The chart below summarizes the compatibility of mounting medium, chromogens and counterstains.

Chromogen Stain Color Enzyme used Solubility in Alcohol/Xylene Compatible with Hematoxylin Compatible Mounting Media
AEC Brick Red HRP Yes Yes Aqueous or Super Mount
DAB Brown HRP No Yes Aqueous, Super Mount or XMount
Elegance Red Red AP No Yes Aqueous, Super Mount or XMount
Fast Red Red AP Yes Yes Aqueous or Super Mount
New Fuchsin Red AP Yes Yes Aqueous or Super Mount