Multispectral LEDs Eliminate Lipofuscin-Associated Autofluorescence for Immunohistochemistry and CD44 Variant Detection by in Situ Hybridization in Aging Human, non-Human Primate, and Murine Brain.

IF 3.9 4区 医学 Q2 NEUROSCIENCES ASN NEURO Pub Date : 2022-01-01 DOI:10.1177/17590914221123138
Philip A Adeniyi, Katie-Anne Fopiano, Fatima Banine, Mariel Garcia, Xi Gong, C Dirk Keene, Larry S Sherman, Zsolt Bagi, Stephen A Back
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Abstract

A major limitation of mechanistic studies in aging brains is the lack of routine methods to robustly visualize and discriminate the cellular distribution of tissue antigens using fluorescent immunohistochemical multi-labeling techniques. Although such approaches are routine in non-aging brains, they are not consistently feasible in the aging brain due to the progressive accumulation of autofluorescent pigments, particularly lipofuscin, which strongly excite and emit over a broad spectral range. Consequently, aging research has relied upon colorimetric antibody techniques, where discrimination of tissue antigens is often challenging. We report the application of a simple, reproducible, and affordable protocol using multispectral light-emitting diodes (mLEDs) exposure for the reduction/elimination of lipofuscin autofluorescence (LAF) in aging brain tissue from humans, non-human primates, and mice. The mLEDs lamp has a broad spectral range that spans from the UV to infrared range and includes spectra in the violet/blue and orange/red. After photo quenching, the LAF level was markedly reduced when the tissue background fluorescence before and after mLEDs exposure was compared (p < 0.0001) across the spectral range. LAF elimination was estimated at 95 ± 1%. This approach permitted robust specific fluorescent immunohistochemical co-visualization of commonly studied antigens in aging brains. We also successfully applied this method to specifically visualize CD44 variant expression in aging human cerebral white matter using RNAscope fluorescent in-situ hybridization. Photo quenching provides an attractive means to accelerate progress in aging research by increasing the number of molecules that can be topologically discriminated by fluorescence detection in brain tissue from normative or pathological aging.

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在衰老的人类、非人灵长类动物和小鼠大脑中,通过原位杂交进行免疫组织化学和 CD44 变异检测时,多光谱 LED 可消除与脂褐素相关的自发荧光。
老化大脑机理研究的一个主要局限是缺乏常规方法,无法利用荧光免疫组化多重标记技术对组织抗原的细胞分布进行有力的观察和鉴别。虽然这种方法在非衰老大脑中是常规方法,但由于自发荧光色素(尤其是脂褐质)的逐渐积累,在衰老大脑中并不总是可行的。因此,衰老研究一直依赖于比色抗体技术,而组织抗原的分辨往往具有挑战性。我们报告了利用多光谱发光二极管(mLEDs)照射来减少/消除人、非人灵长类动物和小鼠衰老脑组织中脂褐素自发荧光(LAF)的一种简单、可重复且经济实惠的方案。mLEDs 灯的光谱范围很广,从紫外到红外,包括紫/蓝和橙/红光谱。光淬灭后,将暴露于 mLEDs 之前和之后的组织背景荧光进行比较,LAF 水平明显降低(p
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来源期刊
ASN NEURO
ASN NEURO NEUROSCIENCES-
CiteScore
7.70
自引率
4.30%
发文量
35
审稿时长
>12 weeks
期刊介绍: ASN NEURO is an open access, peer-reviewed journal uniquely positioned to provide investigators with the most recent advances across the breadth of the cellular and molecular neurosciences. The official journal of the American Society for Neurochemistry, ASN NEURO is dedicated to the promotion, support, and facilitation of communication among cellular and molecular neuroscientists of all specializations.
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