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.
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
- Theodore J Zwang
- Theodore J Zwang
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
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).
- Albert Cardona, University of Cambridge, United Kingdom
- Preprint posted: May 26, 2022 (view preprint)
- Received: October 11, 2022
- Accepted: January 18, 2023
- Accepted Manuscript published: January 19, 2023 (version 1)
- Version of Record published: January 31, 2023 (version 2)
- Version of Record updated: February 10, 2023 (version 3)
- Version of Record updated: March 3, 2023 (version 4)
© 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.
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