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Immunohistochemistry (IHC) is used to detect protein expression in formalin-fixed paraffin-embedded (FFPE) tissue samples. Protein expression can be assessed via chromogenic or fluorescent detection. Both detection systems rely on antigen recognition mediated by a primary antibody. Chromogenic IHC utilizes enzyme-catalyzed deposition of chromogens at the site of the antigen, while Fluorescent IHC employs fluorophores and direct or indirect immunofluorescence to help visualize antigens of interest. While both detection systems allow for some degree of multiplexing (detection of two or more targets), the fluorescent method is particularly amenable to multi-target (up to 6 or more) detection. For further information please visit our Fluorescent Multiplex IHC page.

Validation for IHC: At CST we strive to address all of your experimental needs to ensure reliable and reproducible immunohistochemical staining. We provide:

  • Over 700 antibodies stringently validated for IHC on FFPE tissue, IHC-P recommended at an optimal dilution using specific buffers and protocols, and thoroughly tested on appropriate model systems.
  • Companion products, such as unmasking buffers, diluents, and detection reagents that help to ensure experimental success.
  • SignalSlide® control slides with sections of paraffin-embedded positive and negative cell pellets that either express or are devoid of the target of interest, respectively, to help assess the performance of your antibody.

Technical Support for IHC: Our knowledgeable technical support scientists are a phone call or email away, so you can get the help that you need at the bench.

Fluorescent versus Chromogenic Detection

The table below compares the key features of fluorescent versus chromogenic readouts for IHC.

  Fluorescent Chromogenic
Imaging Platform Confocal or Widefield Fluorescence Microscopy (utilizes filtered light) Brightfield microscopy (uses unfiltered white light)
Resolution Robust for both co-expressed proteins and proteins that do not co-localize Robust for proteins that do not localize to the same subcellular compartment
Autofluoresence Prevalent in violet, blue, and green channels Not applicable
Tissue Architecture Not visible Visible
Multiplex Potential Up to 6 targets or more Up to 4 targets

While fluorescent imaging provides higher resolution and a greater potential to multiplex, the advantage of chromogenic detection is the ability to visualize the framework and morphology of the tissue with the use of brightfield microscopy.

Staining examples with the two detection methods are shown below.

Staining examples with the two detection methods are shown below

Immunohistochemical staining was performed on serial sections of ovarian carcinoma using PD-L1 (E1L3N®) XP® Rabbit mAb #13684 (left), B7-H4 (D1M8I) XP® Rabbit mAb #14572 (middle), and Pan Keratin (C11) Mouse mAb #4545 to evaluate the pattern and level of expression of these two immune checkpoint proteins using fluorescent and chromogenic detection systems. Note: the images taken were of different fields of view.

Both detection systems reveal equivalent detail of protein level and distribution. Thus, the choice of a detection method is entirely dependent on the objective of the experiment.

LKB1 Immunohistochemistry Data

LKB1 Immunohistochemistry Data

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using LKB1 (D60C5F10) Rabbit mAb (IHC Formulated) #13031.

ALK Immunohistochemistry Data

ALK Immunohistochemistry Data

Immunohistochemical analysis of paraffin-embedded anaplastic large cell lymphoma (ALCL) using ALK (D5F3) XP® Rabbit mAb #3633.