Immunocytochemistry (ICC) is often called immunofluorescence (IF) and is characterized by imaging primary cells or cell lines in culture. Immunohistochemistry (IHC) is simply the detection of antibodies in tissue sections, whether it be by chromogenic or fluorescent realization methods. BioLegend products designated as IHC-P indicates the antibody is useful in formalin-fixed paraffin-embedded (FFPE) sections and IHC-F indicates the antibody is only useful in tissue that has been fixed and frozen prior to sectioning. If there is only an IHC designation, check with the literature citations to determine the method of tissue preparation compatible with each reagent.

Chromogenic vs. Fluorescent Imaging Methods

Chromogenic detection methods are advantageous because a signal can be amplified simply by extending the amount of time and substrate in the reaction. Also, it does not require sophisticated instruments for detection, only a microscope with phase contrast. HRP detection can, however, be accompanied by endogenous background associated with cellular peroxidase activity, non-specific signal and is only typically used to image a single marker at a time.



IHC staining of anti-DJ-1 antibody (clone A16125E) on FFPE normal (left) and Parkinson's disease (right) brain tissue.

Fluorescent detection allows visualization of multiple markers at a time, most commonly through the use of discrete excitation sources optimal for each fluorophore. Fluorescent detection introduces the opportunity for advanced imaging applications as well, like live-cell imaging, multiphoton imaging, super-resolution microscopy, FLIM and FRET, just to name a few. Sensitivity can be a limitation of fluorescence microscopy at certain wavelengths, especially reagents that emit in the range of 350-450 nm due to increased autofluorescence of the sample. However, improved signal-to-noise can be obtained through varying enzymatic and immunologic amplification techniques, the use of higher sensitivity instrumentation, and near-infrared emitting fluorophores.

Five-Color Fluorescence Microscopy

With increased fluorophore options and the abundance of directly conjugated antibodies, it is possible to use fluorescence microscopy to look at 5 different markers on a single sample. For larger microscopy panels, it is especially critical to know your microscope and be sure it has the appropriate lasers and filters to capture the emission and excitation spectra of each of these distinct fluorophores.

We stained frozen C57BL/6 mouse spleen tissue using antibodies against CD4 and CD8a to detect T cells, B220 to stain B cells, and CD169 and F4/80 to detect tissue-resident macrophages. For this staining, we took advantage of antibodies directly-conjugated to bright photostable fluorophores including the Brilliant Violet™ and Alexa Fluor® dyes.



Microscope Excitation and Emission Filters

By optimizing your filter set-up to allow for the detection of Brilliant Violet 421™ and Brilliant Violet 510™, you can expand your microscopy panels. See how we optimized our set-up below.



2017 Cell Life Imaging Contest

View all the entry images and discover the ways our customers have used our microscopy reagents in our past Cell Life Imaging Contest.


Grand Prize Winner

Hugues Lelouard – Centre d'Immunologie de Marseille-Luminy

Frozen mouse intestinal villi are stained with anti-EpCAM eFluor™ 450 (cyan), purified anti-Meca 32 (magenta), anti-Lyve 1 eFluor™ 570 (orange), purified anti-CD11c (red), anti-CX3CR1 GFP (green), anti-UEA-I biotin (grey), purified anti-collagen IV (yellow) and Alexa Fluor® 660 phalloidin (purple). The purified antibodies were detected with goat anti-rabbit Alexa Fluor® 514, donkey anti-armenian hamster Alexa Fluor® 594, donkey anti-rat Alexa Fluor® 647 and Streptavidin Alexa Fluor® 405.