IHC Positive Hybridoma Screening Introduction

Immunohistochemistry (IHC) is an antibody-based technique for detecting protein expression in tissues. It is widely used in medical research laboratories as well as clinical settings in order to study the presence of antigens in biological samples. IHC relies on the specific detection of an antibody against a certain epitope, and this specific binding relationship places additional demands on antibody production. In the hybridoma screening process, Creative Biolabs obtains IHC-specific antibodies by performing IHC-positive hybridoma screening. We have a complete and advanced hybridoma platform to provide you with comprehensive hybridoma experimental services.

What Is Immunohistochemistry?

IHC uses the principle of specific binding of antigen and antibody to identify antigens (peptides and proteins) within tissue cells by chemical reactions that cause the color development of chromogenic agents (fluorescein, enzymes, metal ions, and isotopes) that label antibodies. The general steps of immunohistochemistry include cleaning slides, embedding tissue, sectioning, harvesting tissue, dewaxing, antigen repair, serum blocking, primary antibody incubation, secondary antibody incubation, the addition of SABC, the addition of chromogenic agents, re-staining, dehydration, sealing of slides, and finally microscopic observation. The two main categories of specimens for immunohistochemistry are tissue specimens and cellular specimens. Tissue specimens include paraffin sections (the preferred method of specimen preparation) and frozen sections. Paraffin sections require uniform thickness, intact sections, and no folds or knife marks.

Given that there are two methods of IHC, direct and indirect detection, when performing hybridoma production of antibodies, we need to design and identify hybridoma production and positive clones according to the specific situation. In general, direct detection is suitable for studying highly expressed antigens, eliminating the need for additional incubation steps with secondary antibodies while increasing the flexibility of the multicolor experimental design. Indirect detection is applicable to all antigens, and the signal can be further amplified by a variety of methods but requires additional blocking steps and controls.

IHC-Positive Hybridoma Screening

The key to designing IHC experiments is the selection of primary antibodies, as successful immunostaining requires the help of primary antibodies that bind specifically to the target antigen. Therefore, when using hybridomas for antibody production, a specific IHC test protocol needs to be determined, and then a suitable antibody production process needs to be developed to obtain accurate IHC antibodies according to the requirements. The most conclusive demonstration of antibody specificity is the lack of staining in tissues or cells where the target protein has been knocked down, in addition to high signal-to-noise ratios, staining patterns consistent with the known localization of the target protein in control cells or tissues, a lack of staining in tissues or cells known not to express the protein, and only a single band seen in immunoblots. In addition, we generally recommend that IHC-positive clones be identified by ELISA, WB, and other assays. Generally, clones identified by ELISA, WB, and other assays are more likely to be IHC-positive clones.

During the hybridoma screening procedure, multiple influencing factors are frequently required to be considered. Creative Biolabs would like to share our knowledge and experience with you to help you perform IHC-positive hybridoma screening more smoothly.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.

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