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.

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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.

 

 

Fluorophore brightness is not a simple measurement of the properties of the individual fluorophore. There are a number of intrinsic and extrinsic factors that affect the final value of brightness.  The concepts are explained below.

  1. The brightness of a fluorophore is the Extinction Coefficient (EC) x Quantum Yield (QY) in water. That’s the output of the fluor. Let’s call it F.
  2. The antibody has a degree of labeling of the fluorophore; let’s call that DOL. So now brightness is defined as F x DOL.
  3. You may be able to localize more than one antibody per antigen, as is the case when using secondary detection methods. Let’s call that A. So now brightness is A x F x DOL.
  4. But ultimately, the brightness of the antibody is determined against the autofluorescence of the background or cells. Let’s call that AUTO. At shorter emission wavelengths (e.g., the FITC channel), autofluorescence can be a massive detractor from the signal. In the near infrared region of a far-red emitting dye, there is hardly any autofluorescence at all. So now brightness can be defined as A x F x DOL - AUTO
  5. If you’re comparing two different fluors, it is also important how the pixels in the camera/PMT are binned, i.e., the power of lasers exciting two fluors of different excitation wavelengths and the quantum efficiency of the cameras/PMTs at two very different emission intensities. All these considerations are not universal and extremely variant from instrument to instrument and from end-user to end-user.

The 5 variables listed above do not change the actual brightness of the antibody. Rather, they all affect the brightness perceived by the end user and their ability to image their intended target. The stars in the night sky aren’t dim because of the light pollution of the inner city. The star’s brightness doesn’t change; the conditions to image them do.