Lilyquist Jenna, White Kirsten Anne Meyer, Lee Rebecca J, Philips Genevieve K, Hughes Christopher R, Torres Salina M
Department of Internal Medicine, Division of Epidemiology, University of New Mexico, Albuquerque, NM Department of Health Sciences Research, Mayo Clinic, Rochester, MN Cancer Research and Treatment Center Center for HPV Prevention, Department of Pathology, University of New Mexico, Albuquerque, NM.
Medicine (Baltimore). 2017 Apr;96(15):e6432. doi: 10.1097/MD.0000000000006432.
Identification of positive staining is often qualitative and subjective. This is particularly troublesome in pigmented melanoma lesions, because melanin is difficult to distinguish from the brown stain resulting from immunohistochemistry (IHC) using horse radish peroxidase developed with 3,3'-Diaminobenzidine (HRP-DAB). We sought to identify and quantify positive staining, particularly in melanoma lesions. We visualized G-protein coupled estrogen receptor (GPER) expression developed with HRP-DAB and counterstained with Azure B (stains melanin) in melanoma tissue sections (n = 3). Matched sections (n = 3), along with 22 unmatched sections, were stained only with Azure B as a control. Breast tissue (n = 1) was used as a positive HRP-DAB control. Images of the stained tissues were generated using a Nuance Spectral Imaging Camera. Analysis of the images was performed using the Nuance Spectral Imaging software and SlideBook. Data was analyzed using a Kruskal-Wallis one way analysis of variance (ANOVA). We showed that a pigmented melanoma tissue doubly stained with anti-GPER HRP-DAB and Azure B can be unmixed using spectra derived from a matched, Azure B-only section, and an anti-GPER HRP-DAB control. We unmixed each of the melanoma lesions using each of the Azure B spectra, evaluated the mean intensity of positive staining, and examined the distribution of the mean intensities (P = .73; Kruskal-Wallis). These results suggest that this method does not require a matched Azure B-only stained control tissue for every melanoma lesion, allowing precious tissues to be conserved for other studies. Importantly, this quantification method reduces the subjectivity of protein expression analysis, and provides a valuable tool for accurate evaluation, particularly for pigmented tissues.
阳性染色的鉴定通常是定性的且主观的。这在色素性黑色素瘤病变中尤其麻烦，因为黑色素很难与使用3,3'-二氨基联苯胺（HRP-DAB）显色的免疫组织化学（IHC）产生的棕色染色区分开来。我们试图鉴定并量化阳性染色，特别是在黑色素瘤病变中。我们在黑色素瘤组织切片（n = 3）中观察了用HRP-DAB显色并用天青B（染黑色素）复染的G蛋白偶联雌激素受体（GPER）表达。匹配的切片（n = 3）以及22个不匹配的切片仅用天青B染色作为对照。乳腺组织（n = 1）用作HRP-DAB阳性对照。使用Nuance光谱成像相机生成染色组织的图像。使用Nuance光谱成像软件和SlideBook对图像进行分析。数据使用Kruskal-Wallis单因素方差分析（ANOVA）进行分析。我们表明，用抗GPER HRP-DAB和天青B双重染色的色素性黑色素瘤组织可以使用来自匹配的、仅用天青B染色的切片和抗GPER HRP-DAB对照的光谱进行解混。我们使用每个天青B光谱对每个黑色素瘤病变进行解混，评估阳性染色的平均强度，并检查平均强度的分布（P = 0.73；Kruskal-Wallis）。这些结果表明，该方法不需要为每个黑色素瘤病变都设置匹配的仅用天青B染色的对照组织，从而可以将珍贵的组织保留用于其他研究。重要的是，这种定量方法减少了蛋白质表达分析的主观性，并为准确评估提供了有价值的工具，特别是对于色素性组织。
