Principles of deep immunohistochemistry for 3D histology - PubMed (original) (raw)
Review
Principles of deep immunohistochemistry for 3D histology
Chun Ngo Yau et al. Cell Rep Methods. 2023.
Abstract
Deep immunohistochemistry (IHC) is a nascent field in three-dimensional (3D) histology that seeks to achieve thorough, homogeneous, and specific staining of intact tissues for visualization of microscopic architectures and molecular compositions at large spatial scales. Despite the tremendous potential of deep IHC in revealing molecule-structure-function relationships in biology and establishing diagnostic and prognostic features for pathological samples in clinical practice, the complexities and variations in methodologies may hinder its use by interested users. We provide a unified framework of deep immunostaining techniques by discussing the theoretical considerations of the physicochemical processes involved, summarizing the principles applied in contemporary methods, advocating a standardized benchmarking scheme, and highlighting unaddressed issues and future directions. By providing the essential information to guide investigators in customizing immunolabeling pipelines, we also seek to facilitate the adoption of deep IHC for researchers to address a wide range of research questions.
© 2023 The Author(s).
Conflict of interest statement
The authors declare no competing interests.
Figures
Graphical abstract
Figure 1
An overview of the RDA process in deep immunostaining (A) Diffusion and advective transport of Abs into the tissue. The effective path from Abs to Ags through cell membranes and the ECM forms the main diffusion barrier (curved arrows), while binding reactions with peripheral Ags form the main reaction barrier (straight arrow). There are two major routes for Abs in the staining buffer (blue) to reach the intra- and extracellular Ags: (i) directly diffusing from the tissue surface and (ii) via the low-resistance tissue vasculature (if intact and patent). Both require diffusion through the dense tissue matrix (pink). The reaction barrier for route (ii) is omitted for simplicity. (B) Penetration of the cell membrane to access intracellular Ags. Without permeabilization, the intact cell membrane acts as a significant barrier to hinder Ab access to intracellular Ags. Extracellular Ags are omitted for simplicity. (C) Ab-Ag reaction and its determinants. Ab-Ag reaction is largely determined by the properties of the local environment, such as temperature, pH, and ionic strength. Alterations of these parameters can affect the non-covalent Ab-Ag interactions. The figure was created with
BioRender
.
Figure 2
A graphical summary of deep immunostaining technical approaches (A) Infusion of Abs into the tissue via the intact vasculature as in vDISCO, which (i) decreases the diffusion distance (_r_1 from the nearest vessel < _r_2 from the tissue surface) and (ii) allows advective transport across vessel walls driven by mechanical pressure gradients to assist Abs to reach deep-tissue Ags. (B) Creation of advection via an electric field utilizing the electromobility of Abs as in SE. The representation of the electric field (purple) here is simplified. (C) Transformation of tissue into a hydrogel, which allows shrinkage in size to reduce diffusion distance, as in ELAST. (D) Optimized permeabilization of the cell membrane to enhance Ab penetration, as in iDISCO and SHANEL, by formation of lipid micelles. Green, membrane lipids; red, detergents. (E) Non-covalent interference of Ab-Ag interactions by manipulation of ionic strength and/or pH (top panel, using NaCl as an example), as in eFLASH, and by addition of detergents (bottom panel), as in SWITCH. Gray, detergents. (F) Thermal inhibition utilizing the exothermic nature of Ab-Ag reaction, as in ThICK with SPEARs. The figure was created with
BioRender
.
Figure 3
A standardized benchmarking scheme for deep IHC In this example, an appropriate marker in a mouse hemibrain is first stained with the deep IHC method of interest (magenta, bulk staining), followed by cutting the tissue and re-staining in standard immunostaining buffer (e.g., PBS with 0.1% Tween 20) for the same marker with a spectrally non-overlapping fluorophore (green, cut staining). The tissue is then imaged on the cut plane, where cross-comparison of both channels will reveal how well the deep IHC method reproduces the expected pattern in section-based conventional IHC. In this illustrated example, the deep immunostaining method has limited penetration, causing a rim of stronger magenta signal near the tissue surface than at its core. Adapted from Lai et al_._
References
- Delesse M. Procédé mecanique pour determiner la composition des roches. CR Acad. Sci. Paris. 1847;25:544–545.
- Spalteholz W. 1911. Über das Durchsightigmachen von menschlichen und tierischen Präparaten: nebst Anhang, Über Knochenfärbung (S. Hirzel)
- Feldkamp L.A., Davis L.C., Kress J.W. Practical cone-beam algorithm. Josa a. 1984;1:612–619.
- Feldkamp L.A., Goldstein S.A., Parfitt A.M., Jesion G., Kleerekoper M. The direct examination of three-dimensional bone architecture in vitro by computed tomography. J. Bone Miner. Res. 1989;4:3–11. -PubMed
Publication types
MeSH terms
LinkOut - more resources
Full Text Sources