Immunohistochemistry (IHC) is an antibody-based technique that detects antigens of interest in fixed tissue sections, often visualized by light microscopy (see figure below). In this multi-step application, staining is highly influenced by multiple variables. High-quality reagents and optimized protocols are key for generating accurate expression, localization and distribution data. Our complete line of IHC products and scientific resources will help you produce valid, reproducible results you can trust.
IHC Detection Methods. As illustrated above, enzymatic conversion of a chromogen substrate is required to visualize an antigen in traditional IHC experiments. Fluorescence detection is an alternative method that is more suitable for multicolor experiments due to the broad availability of fluorochromes and the emergence of high contrast imaging.
Read more about IHC detection methods.
IHC Primary Antibodies
The most critical factor to achieve successful staining is selecting an antibody that specifically binds the target antigen. Novus offers over 34,000 IHC validated antibodies referenced in nearly 7,000 peer-reviewed publications that can be tested risk free with the Novus Quality Guarantee. Read more.
In indirect detection, a labeled secondary antibody (fluorescent, enzymatic, biotin, etc.) and, in some cases, a third layer for signal amplification is required to detect the antigen-primary antibody complex. Compared to directly labeled primary antibodies, indirect detection is more sensitive and vital for effective identification of low abundance antigens, rare epitopes, and under conditions of nonoptimal antigen-antibody binding. Learn more about secondary antibodies.
Due to the number of variables that impact staining, including the proper controls in IHC experiments such as tissue controls are important for identifying the source of staining issues and for validating results. Recommendations to verify the specificity of antigen-antibody interactions include absorption controls involving blocking peptides and isotype controls, which are used in place of primary antibodies.
Moving Beyond Protein Expression - Dual in situ Hybridization (ISH)/IHC
Combining the protein detection capabilities of IHC with single-molecule RNA expression analysis using RNAScope® ISH technology builds a more complete and robust picture of the molecular mechanisms involved in numerous biological pathways. View our recorded webinar on applying dual IHC/ISH methods below.