Tissue libraries enable rapid determination of conditions that preserve antibody labeling in cleared mouse and human tissue

  1. Theodore J Zwang  Is a corresponding author
  2. Rachel E Bennett
  3. Maria Lysandrou
  4. Benjamin Woost
  5. Anqi Zhang
  6. Charles M Lieber
  7. Douglas S Richardson
  8. Bradley T Hyman
  1. Massachusetts General Hospital, United States
  2. Stanford University, United States
  3. Harvard University, United States

Abstract

Difficulty achieving complete, specific, and homogenous staining is a major bottleneck preventing the widespread use of tissue clearing techniques to image large volumes of human tissue. In this manuscript, we describe a procedure to rapidly design immunostaining protocols for antibody labeling of cleared brain tissue. We prepared libraries of .5-1.0 mm thick tissue sections that are fixed, pre-treated, and cleared via similar, but different procedures to optimize staining conditions for a panel of antibodies. Results from a library of mouse tissue correlate well with results from a similarly prepared library of human brain tissue, suggesting mouse tissue is an adequate substitute for protocol optimization. These data show that procedural differences do not influence every antibody-antigen pair in the same way, and minor changes can have deleterious effects, therefore, optimization should be conducted for each target. The approach outlined here will help guide researchers to successfully label a variety of targets, thus removing a major hurdle to accessing the rich 3D information available in large, cleared human tissue volumes.

Data availability

All data used in publication can be accessed at the BioImage Archive https://www.ebi.ac.uk/biostudies/studies/S-BIAD479All Matlab code and ImageJ macros used to quantify data can be accessed at https://github.com/tjzwang/IHC

The following data sets were generated

Article and author information

Author details

  1. Theodore J Zwang

    Department of Neurology, Massachusetts General Hospital, Boston, United States
    For correspondence
    tzwang@mgh.harvard.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6815-4482
  2. Rachel E Bennett

    Department of Neurology, Massachusetts General Hospital, Boston, United States
    Competing interests
    No competing interests declared.
  3. Maria Lysandrou

    Department of Neurology, Massachusetts General Hospital, Boston, United States
    Competing interests
    No competing interests declared.
  4. Benjamin Woost

    Department of Neurology, Massachusetts General Hospital, Boston, United States
    Competing interests
    No competing interests declared.
  5. Anqi Zhang

    Department of Chemical Engineering, Stanford University, Stanford, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6121-8095
  6. Charles M Lieber

    Department of Chemistry and Chemical Biology, Harvard University, Cambridge, United States
    Competing interests
    No competing interests declared.
  7. Douglas S Richardson

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3189-2190
  8. Bradley T Hyman

    Department of Neurology, Massachusetts General Hospital, Boston, United States
    Competing interests
    Bradley T Hyman, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7959-9401

Funding

National Institute on Aging (NIA-K99AG068602)

  • Theodore J Zwang

Massachusetts General Hospital (Harrison Gardner,Jr Innovation Award)

  • Theodore J Zwang

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the mice were handled according to approved institutional animal care and use committee (IACUC protocol # 2019N000026 and 2020N000069).

Human subjects: Autopsy tissue from human brains were collected at Massachusetts General hospital, with informed consent of patients or their relatives and approval of local institutional review boards. Human tissue was provided by the Massachusetts Alzheimer's Disease Research Center (ADRC) with approval from the Mass General Brigham IRB (1999P009556).

Copyright

© 2023, Zwang et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,436
    views
  • 225
    downloads
  • 4
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Theodore J Zwang
  2. Rachel E Bennett
  3. Maria Lysandrou
  4. Benjamin Woost
  5. Anqi Zhang
  6. Charles M Lieber
  7. Douglas S Richardson
  8. Bradley T Hyman
(2023)
Tissue libraries enable rapid determination of conditions that preserve antibody labeling in cleared mouse and human tissue
eLife 12:e84112.
https://doi.org/10.7554/eLife.84112

Share this article

https://doi.org/10.7554/eLife.84112

Further reading

    1. Cell Biology
    2. Stem Cells and Regenerative Medicine
    Nathaniel Paul Meyer, Tania Singh ... Diane L Barber
    Research Article

    Our understanding of the transitions of human embryonic stem cells between distinct stages of pluripotency relies predominantly on regulation by transcriptional and epigenetic programs with limited insight on the role of established morphological changes. We report remodeling of the actin cytoskeleton of human embryonic stem cells (hESCs) as they transition from primed to naïve pluripotency which includes assembly of a ring of contractile actin filaments encapsulating colonies of naïve hESCs. Activity of the Arp2/3 complex is required for the actin ring, to establish uniform cell mechanics within naïve colonies, promote nuclear translocation of the Hippo pathway effectors YAP and TAZ, and effective transition to naïve pluripotency. RNA-sequencing analysis confirms that Arp2/3 complex activity regulates Hippo signaling in hESCs, and impaired naïve pluripotency with inhibited Arp2/3 complex activity is rescued by expressing a constitutively active, nuclear-localized YAP-S127A. Moreover, expression of YAP-S127A partially restores the actin filament fence with Arp2/3 complex inhibition, suggesting that actin filament remodeling is both upstream and downstream of YAP activity. These new findings on the cell biology of hESCs reveal a mechanism for cytoskeletal dynamics coordinating cell mechanics to regulate gene expression and facilitate transitions between pluripotency states.

    1. Cell Biology
    Eleanor Martin, Rossana Girardello ... Alexander Ludwig
    Research Article

    Caveolae are small membrane pits with fundamental roles in mechanotransduction. Several studies have shown that caveolae flatten out in response to an increase in membrane tension, thereby acting as a mechanosensitive membrane reservoir that buffers acute mechanical stress. The dynamic assembly and disassembly of caveolae has also been implicated in the control of RhoA/ROCK-mediated actomyosin contractility at the rear of migrating cells. However, how membrane tension controls the organisation of caveolae and caveolae-mediated mechanotransduction is poorly understood. To address this, we systematically quantified protein-protein interactions of caveolin-1 in migrating RPE1 cells at steady state and in response to an acute increase in membrane tension using biotin-based proximity labelling and quantitative mass spectrometry. Our data show that caveolae are highly enriched at the rear of migrating RPE1 cells and that membrane tension rapidly and reversibly disassembles the caveolar protein coat. Membrane tension also dislodges caveolin-1 from focal adhesion proteins and several mechanosensitive cortical actin regulators including filamins and cortactin. In addition, we present evidence that ROCK and the RhoGAP ARHGAP29 are associated with caveolin-1 in a membrane tension-dependent manner, and that ARHGAP29 regulates caveolin-1 Y14 phosphorylation, caveolae rear localisation, and RPE1 cell migration. Taken together, our work uncovers a membrane tension-sensitive coupling between caveolae and the rear-localised F-actin cytoskeleton. This provides a framework for dissecting the molecular mechanisms underlying caveolae-regulated mechanotransduction pathways.