High-contrast enzymatic immunohistochemistry of pigmented tissues


  • Sara M. Duncan University at Buffalo
  • Gail M. Seigel University at Buffalo




Enzyme immunohistochemistry, pigmented tissue, peroxidase substrate, VIP purple substrate, diaminobenzidine, retinal pigmented epithelial cells


Historically, standard enzyme immunohistochemistry has been accomplished with brown (DAB, diaminobenzidine) substrate. This can become problematic in pigmented tissues, such as the retina, where brown pigment of retinal pigmented epithelial (RPE) cells can be easily confounded with brown substrate.  Although immunofluorescence detection methods can overcome this challenge, fluorescence may fade over a period of weeks, while enzyme substrates allow for more long-lasting, archival results. In this report, we will describe a high-contrast enzyme immunohistochemistry method ideal for pigmented tissues that utilizes purple (VIP) substrate. We compared brown (DAB) and purple (VIP) substrates in enzyme immunohistochemistry experiments using human retina (paraffin sections) and monkey retinal pigmented epithelial cells (frozen sections), both containing brown pigmented cells. We compared substrates using several primary antibodies against markers that can be detected in the retina, including GFAP, VEGF, CD147 (EMMPRIN), RHO (rhodopsin) and PAX6.  Methyl green was used as a counterstain for paraffin sections. A side-by-side comparison between DAB and VIP immunohistochemistry showed excellent contrast between pigmented cells and the purple VIP substrate in both human retinal tissue and monkey pigmented epithelial cells for all of the markers tested. This was a marked improvement over DAB staining in pigmented cells and tissues. For both paraffin sections and frozen sections of pigmented tissues, purple VIP substrate is an excellent alternative to brown DAB substrate and non-permanent immunofluorescence methods.


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How to Cite

Duncan SM, Seigel GM. High-contrast enzymatic immunohistochemistry of pigmented tissues. J Biol Methods [Internet]. 2016Jul.12 [cited 2021Dec.4];3(3):e47. Available from: https://jbmethods.org/jbm/article/view/122