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Protocol for multiplex fluorescent immunohistochemistry in free-floating rodent brain tissues.

ABSTRACT: Identifying multiple proteins within the same tissue allows for assessing protein colocalization, is cost effective, and maximizes efficiency. Here, we describe a protocol for multiplex immunolabeling of proteins in free-floating rodent brain sections. As opposed to slide-mounted immunohistochemistry, the free-floating approach results in less tissue loss and greater antibody penetration. Using distinct fluorophores for individual proteins, this protocol allows for visualization of three or more proteins within tissue sections. The protocol can be applied to other tissue types. For complete details on the use and execution of this protocol, please refer to Gonzalez Abreu et al. (2022).

SUBMITTER: Kelly AM

PROVIDER: S-EPMC9483644 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Protocol for multiplex fluorescent immunohistochemistry in free-floating rodent brain tissues.

Kelly Aubrey M AM Fricker Brandon A BA Wallace Kelly J KJ

STAR protocols 20220914 4

Identifying multiple proteins within the same tissue allows for assessing protein colocalization, is cost effective, and maximizes efficiency. Here, we describe a protocol for multiplex immunolabeling of proteins in free-floating rodent brain sections. As opposed to slide-mounted immunohistochemistry, the free-floating approach results in less tissue loss and greater antibody penetration. Using distinct fluorophores for individual proteins, this protocol allows for visualization of three or more ...[more]

PMID: 36107743

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Project description:BackgroundAstrocytes and microglia react to Aβ plaques, neurofibrillary tangles, and neurodegeneration in the Alzheimer's disease (AD) brain. Single-nuclei and single-cell RNA-seq have revealed multiple states or subpopulations of these glial cells but lack spatial information. We have developed a methodology of cyclic multiplex fluorescent immunohistochemistry on human postmortem brains and image analysis that enables a comprehensive morphological quantitative characterization of astrocytes and microglia in the context of their spatial relationships with plaques and tangles.MethodsSingle FFPE sections from the temporal association cortex of control and AD subjects were subjected to 8 cycles of multiplex fluorescent immunohistochemistry, including 7 astroglial, 6 microglial, 1 neuronal, Aβ, and phospho-tau markers. Our analysis pipeline consisted of: (1) image alignment across cycles; (2) background subtraction; (3) manual annotation of 5172 ALDH1L1+ astrocytic and 6226 IBA1+ microglial profiles; (4) local thresholding and segmentation of profiles; (5) machine learning on marker intensity data; and (6) deep learning on image features.ResultsSpectral clustering identified three phenotypes of astrocytes and microglia, which we termed "homeostatic," "intermediate," and "reactive." Reactive and, to a lesser extent, intermediate astrocytes and microglia were closely associated with AD pathology (≤ 50 µm). Compared to homeostatic, reactive astrocytes contained substantially higher GFAP and YKL-40, modestly elevated vimentin and TSPO as well as EAAT1, and reduced GS. Intermediate astrocytes had markedly increased EAAT2, moderately increased GS, and intermediate GFAP and YKL-40 levels. Relative to homeostatic, reactive microglia showed increased expression of all markers (CD68, ferritin, MHC2, TMEM119, TSPO), whereas intermediate microglia exhibited increased ferritin and TMEM119 as well as intermediate CD68 levels. Machine learning models applied on either high-plex signal intensity data (gradient boosting machines) or directly on image features (convolutional neural networks) accurately discriminated control vs. AD diagnoses at the single-cell level.ConclusionsCyclic multiplex fluorescent immunohistochemistry combined with machine learning models holds promise to advance our understanding of the complexity and heterogeneity of glial responses as well as inform transcriptomics studies. Three distinct phenotypes emerged with our combination of markers, thus expanding the classic binary "homeostatic vs. reactive" classification to a third state, which could represent "transitional" or "resilient" glia.

Dataset Information

Protocol for multiplex fluorescent immunohistochemistry in free-floating rodent brain tissues.

Publications

Protocol for multiplex fluorescent immunohistochemistry in free-floating rodent brain tissues.

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