Secondary Antibody Handbook
Counterstaining and Mounting ICC IF
The Difference Between Immunocytochemistry (ICC), Immunohistochemistry (IHC), and Immunofluorescence (IF)
Immunocytochemistry (ICC), Immunohistochemistry (IHC) and Immunofluorescence (IF) all utilize antibodies to provide visual details about protein abundance, distribution, and localization. These terms are often confusing and are sometimes mistakenly used interchangeably. Thus, it is important to understand the fundamental differences between these various techniques.
IHC-Fr (left): Cadherin‑17 was detected in perfusion fixed frozen sections of mouse intestine using rabbit anti-mouse Cadherin‑17 monoclonal antibody (Catalog # MAB8524), followed by the NorthernLights™ 557-conjugated anti-rabbit IgG secondary antibody (red; Catalog # NL004) and counterstained with DAPI (blue). Specific staining of Cadherin-17 was localized to plasma membranes. ICC/IF (right): E‑Cadherin was detected in immersion fixed D3 mouse embryonic stem cell line using goat anti-Mouse E‑Cadherin Antigen Polyclonal Antibody (Catalog # AF748) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated anti-goat IgG secondary antibody (red; Catalog # NL001) and counterstained with DAPI (blue). Specific staining was localized to cell surfaces.
What Samples are Used in ICC vs IHC?
Sample Type and Preparation - tissue vs cells – As the name implies, IHC detection refers to tissue immunostaining, of either formalin fixed paraffin-embedded (FFPE) or frozen tissue. ICC refers to the staining of isolated or cultured intact cells where samples may be from tissue culture cell lines, either adherent or in suspension. Prior to staining, IHC and ICC samples are also processed differently. A few key differences between applications include:
What Labeling Methods are Best for ICC and IHC?
Labeling Method – chromogenic vs fluorescent – IHC and ICC have traditionally used chromogenic reagents to detect target antigens. In chromogenic detection, an enzyme such as horseradish peroxidase (HRP) converts a soluble substrate including 3,3'-Diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC) into an insoluble colored product at the antigen site. In IF detection, the fluorochrome reagent labels, either directly or indirectly, the primary antibody and, once excited, will emit light at a specific wavelength.
With the extensive list of available fluorescent labels and their ease of use in multiplex applications, researchers are choosing ICC/IF and fluorescent IHC more regularly. Additionally, the use of fluorescent conjugated antibodies specific against organelle markers, facilitates the analysis of proteins of interest and extends multiplex capabilities in both IHC and ICC. For a comprehensive comparison of chromogenic and fluorescence detection methods, review the IHC Detection Guide.
Looking for a validated secondary antibody to detect your antigen of interest? Novus Biologicals offers thousands of conjugated antibodies including more than 40 different enzymatic, fluorescent, or biotin labels to choose from.