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