High-contrast enzymatic immunohistochemistry of pigmented tissues

Authors

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

DOI:

https://doi.org/10.14440/jbm.2016.122

Keywords:

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

Abstract

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.

References

De Matos, LL, Trufelli, DC, de Matos, MGL, da Silva Pinhal, MA (2010) Immunohistochemistry as an important tool in biomarker detection and clinical practice. Biomarker Insights 5: 9–20.

Talve LA, Collan YU, Alanen KA. (1995) Bleaching of melanin before image cytometry of the DNA content of pigmented skin tumors. Anal Quant Cytol Histol 17(5): 344-50.

Kligora CJ1, Fair KP, Clem MS, Patterson JW (1999) A comparison of melanin bleaching and azure blue counterstaining in the immunohistochemical diagnosis of malignant melanoma. Mod Pathol 12(12): 1143-7.

Marmorstein LY, Munier FL, Arsenijevic Y, Schorderet DF, McLaughlin PJ, Chung D, Traboulsi E, and Marmorstein AD (2002) Aberrant accumulation of EFEMP1 underlies drusenformation in Malattia Leventinese and age-related macular degeneration Proc Natl Acad Sci U S A 99(20): 13067–13072.

Omarova S, Charvet CD, Reem RE, Mast N, Zheng W, Huang S, Peachey NS, Pikuleva IA (2012) Abnormal vascularization in mouse retina with dysregulated retinal cholesterol homeostasis. J Clin Invest 122(8): 3012-3023.

Liu RT, Gao J, Cao S, Sandhu N, Cui JZ, Chou CL Fang E, Matsubara JA (2013) Inflammatory mediators induced by amyloid-beta in the retina and RPE in vivo: Implications for inflammasome activation in age-related macular degeneration. Investigative Ophthalmology & Visual Science 54: 2225-2237.

Pfeffer BA (1990) Improved methodology for cell culture of human and monkey retinal pigment epithelium. In: Progress in Retinal Research (N.N. Osborne and G.J. Chader, eds.), Volume 10, pp. 251-291. Pergamon Press, Oxford, U.K.

Pfeffer BA, Becerra SP, Borst DE, and Wong P (2004) Expression of transthyretin and retinol binding protein mRNAs and secretion of transthyretin by cultured monkey retinal pigment epithelium. Mol Vis 10: 23-30.

Becerra SP, Fariss RN, Wu YQ, Montuenga LM, Wong P, and Pfeffer BA (2004) Pigment epithelium-derived factor in the monkey retinal pigment epithelium and interphotoreceptor space: apical secretion and distribution. Exp Eye Res 78(2): 223-234.

Ishihara, N., Jofuku, A., Eura, Y., Mihara, K., (2003) Regulation of mitochondrial morphology by membrane potential, and DRP1-dependent division and FZO1-dependent fusion reaction in mammalian cells. Biochem. Biophys. Res. Comm. 301: 891-898.

Zhou, M. and Grofova, I (1995) The use of peroxidase substrate Vector® VIP in electron microscopic single and double antigen localization. J. Neurosci. Methods 62: 149–158.

Lanciego JL, Goede PH, Witter MP, Wouterlood FG (1997) Use of peroxidase substrate Vector VIP for multiple staining in light microscopy. J Neurosci Methods 74(1): 1-7.

Kliffen M, Sharma HS, Mooy CM, Kerkvliet S, de Jong PT (1997) Increased expression of angiogenic growth factors in age-related maculopathy. Br J Ophthalmol 81(2): 154-62.

Ford KM, D'Amore PA (2012) Molecular regulation of vascular endothelial growth factor expression in the retinal pigment epithelium. Mol Vis 18: 519-27. Epub 2012 Mar 1.

Zhao TT1, Tian CY, Yin ZQ (2010) Activation of Müller cells occurs during retinal degeneration in RCS rats. Adv Exp Med Biol 664: 575-83.

Malanson KM, Lem J (2009) Rhodopsin-mediated retinitis pigmentosa. Prog Mol Biol Transl Sci 88:1–31.

Shah SP, Wong F, Sharp DM, Vincent AL (2014) A novel rhodopsin point mutation, proline-170-histidine, associated with sectoral retinitis pigmentosa. Ophthalmic Genet 35(4):241-7.

Marmorstein AD1, Gan YC, Bonilha VL, Finnemann SC, Csaky KG, Rodriguez-Boulan E (1998) Apical polarity of N-CAM and EMMPRIN in retinal pigment epithelium resulting from suppression of basolateral signal recognition. J Cell Biol 142(3): 697-7.

Raviv S, Bharti K, Rencus-Lazar S, Cohen-Tayar Y, Schyr R, Evantal N, Meshorer E, Zilberberg A, Idelson M, Reubinoff B, Grebe R, Rosin-Arbesfeld R, Lauderdale J, Lutty G, Arnheiter H, Ashery-Padan R (2014) PAX6 regulates melanogenesis in the retinal pigmented epithelium through feed-forward regulatory interactions with MITF. PLoS Genet 10(5): e1004360.

Namimatsu S, Ghazizadeh M and Sugisaki Y (2005) Reversing the effects of formalin fixation with citraconic anhydride and heat: a universal antigen retrieval method. J Histochem Cytochem. 53(1):3-11.

Liu CH, Lin CH, Tsai MJ, Chen WT, Chai CY, Huang YC, Tsai KB. (2003) Melanin bleaching with dilute hydrogen peroxide: a simple and rapid method. Appl Immunohistochem Mol Morphol. May;21(3):275-9.

Manicam C, Pitz S, Brochhausen C, Grus FH, Pfeiffer N, Gericke A. (2014) Effective melanin depigmentation of human and murine ocular tissues: an improved method for paraffin and frozen sections. PLoS One. Jul 15;9(7):e102512.

Downloads

Published

2016-07-12

How to Cite

1.
Duncan SM, Seigel GM. High-contrast enzymatic immunohistochemistry of pigmented tissues. J Biol Methods [Internet]. 2016Jul.12 [cited 2021May8];3(3):e47. Available from: https://jbmethods.org/jbm/article/view/122

Issue

Section

Articles