Immunohistology buffers

Low background and strong specific reaction. Standardized staining.

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Any antibody preparation has some potential to produce non-specific reaction in the assay. This originates from:

  • non-specific antibodies that are present in some proportion in any polyclonal antibody preparation, including affinity purified ones (often "affinity purified" means only isolation of IgG fraction on Prot A/G column, not the purification on the antigen column under very stringent conditions)

  • low specificity antibodies among specific ones in polyclonal

  • fragments of fallen apart IgGs in stored preparations, including monoclonal

  • separate heavy and light chains of specific antibodies, produced by most hybridomas

 

All these are capable of binding non-specifically to molecules on tissue sections, blots, fixed cells and other objects for immune detection. In case of retrieved formalin sections the risk of non-specific reaction is even higher, since to activate the epitope recovery the proteins comprising the tissue sections are denatured during HIER, thus making many domains accessible that are charged and capable of binding the test immunoglobulins in a non-specific manner.

 

The standard means to block non-specific binding of specific antibody preparation is to add an irrelevant protein, such as BSA, other serum, casein, etc. However, everyone who tried to do this knows that increasing (for effective blocking) concentration of such blocking agent leads to a great reduction of the specific reaction as well. This is due to large blocking molecules binding to accessible sites on section and thus sterically blocking access of specific antibodies to epitopes of interest (schematically represented in the figure on the left, top). All our buffers developed for immune assays isntead contain short (0.6-2 kD) peptides that are capable of effectively blocking non-specific reactions while not affecting the specific binding of antibody.(fugure on the left, bottom)

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Aptum's collection of Immunohistology buffers has also some other benefits (see below) and allows you to achieve the best quality IHC result without compromising the antigen detection. The buffers can also be used in other immune assays, such as immunofluorescence on sections, flow cytometry on fixed cells, hybridization of sections with antibody detection.

 

The Retriever IHC buffers empower you to control non-specific staining on every step of immunohistochemistry. They are especially highly recommended for research pathology where, in contrast to diagnostics, many polyclonal and/or low-affinity antibodies are used.

 

Section Block 

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Blocking solution based on chemically modified and fragmented ultra-pure casein. Effectively reduces unwanted binding of primary antibody and conjugates you use to a charged surface of the slide and tissue section. Greatly reduces non-specific binding while preserving the specific reaction. Saturates potential non-specific protein-protein interactions. In contrast to BSA-based, IgGs, casein, or serum -based blocking solutions there is no interaction of specific antibody and blocking protein itself.  Recommended for research and diagnostic pathology, especially for retrieved sections and polyclonal antibodies. 

Antibody Diluent - P

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Buffer for diluting your primary and secondary antibodies, especially if they planned to be stored for a while in the refrigerator. Nonspecific binding and low or medium affinity cross-reactivities of the antibodies will be minimised, making your result more reliable. Excellent for IHC on formalin-fixed retrieved and non-retrieved sections. Great for flow cytometry on fixed cells. When used in pathology, in also greatly reduces non-specific reactivity of human serum components and immunoglobulins in tissue, vessels and cells with mouse antibodies used in the section. 

For especially "trouble"-giving antibodies, as well as for in situ PCR applications, this diluent may also be used as a washing buffer, preventing secondary binding of your antibodies during washing.

Antibody Diluent-F

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Similar to Antibody Diluent-P, but specially designed for frozen (cryostat) sections. Greatly reduces non-specific binding while preserving the specific reaction, by saturating potential non-specific protein-protein interactions. 

Peroxidase Conjugate Diluent

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Specifically designed for preparing the solution of your HRP-conjugate used as the detection reagent. It is the Antibody-diluent buffer with additional component for stabilising your HRP-conjugate. Allows you to further standardise the assay preparing ready-to-use conjugate solutions in advance and store them in refrigerator without loss of activity.

Slides Washing Buffer PBS*

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Ready-to-use buffer to wash slides with tissue sections during IHC experiments. Recommended for rinzing slides after blocking buffer (up to 5 min) and after primary antibody and conjugate. Recommended for retrieved FF/PE tissues, wholemounts.

Slides Washing Buffer Tris*

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Ready-to-use buffer to wash slides with tissue sections during IHC experiments. Recommended for rinzing slides after blocking buffer (up to 5 min) and after primary antibody and conjugate. Recommended for retrieved FF/PE tissues, wholemounts. 

* Please see the IHC Blog Note on relative advantages of using Tris- or PBS buffers.

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