Enhanced tissue penetration of antibodies through pressurized immunohistochemistry

Fiorelli, Roberto; Sidhu, Gurpaul S.; Cebrián-Silla, Arantxa; Melendez, Ernesto Luna; Mehta, Shwetal; García‐Verdugo, José Manuel; Sanai, Nader

doi:10.1101/2020.09.25.311936

Cited by 7 publications

(8 citation statements)

References 43 publications

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“…There is also the separate issue of obtaining homogeneous and consistent antibody or fluorophore staining through thick cleared tissue. Traditionally, poor antibody penetration has been combatted with drastically increased incubation times, or more recently with stochastic electrotransport ( Kim et al., 2015 ) and barometric pressure ( Fiorelli et al., 2020 ). While these solutions have resulted in better efficacy of immunolabeling, increased incubation times increased the risk of microbial growth during the staining process, and techniques such as stochastic electrotransport may not be easily implemented into current laboratory workflows.…”

Section: Introductionmentioning

confidence: 99%

Visualization and analysis of whole depot adipose tissue neural innervation

et al. 2021

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Summary Little is known about the diversity and function of adipose tissue nerves, due in part to the inability to effectively visualize the tissue’s diverse nerve subtypes and the patterns of innervation across an intact depot. The tools to image and quantify adipose tissue innervation are currently limited. Here, we present a method of tissue processing that decreases tissue thickness in the z-axis while leaving cells intact for subsequent immunostaining. This was combined with autofluorescence quenching techniques to permit intact whole tissues to be mounted on slides and imaged by confocal microscopy, with a complementary means to perform whole tissue neurite density quantification after capture of tiled z-stack images. Additionally, we demonstrate how to visualize nerve terminals (the neuro-adipose nexus) in intact blocks of adipose tissue without z-depth reduction. We have included examples of data demonstrating nerve subtypes, neurovascular interactions, label-free imaging of collagen, and nerve bundle digital cross-sections.

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Section: Introductionmentioning

confidence: 99%

Visualization and analysis of whole depot adipose tissue neural innervation

et al. 2021

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“…However, along with sample preparation, immunostaining also takes a majority of assay time and cost as it often requires hours or even overnight antibody incubation, and a single vial of antibody often costs a few hundred dollars. Several attempts have been made to reduce the incubation time and antibody consumption volumes by applying electrical fields, barometric pressure, microfluidic structures, ,, or magnetic beads . Unfortunately, these methods are not readily compatible with clinical cellular and tissue specimens and require special instruments.…”

Section: Conclusion and Discussionmentioning

confidence: 99%

Hydrogel Stamping for Rapid, Multiplexed, Point-of-Care Immunostaining of Cells and Tissues

Chin

Choi³

et al. 2022

ACS Appl. Mater. Interfaces

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In the era of precision oncology, multicolor fluorescence imaging has become a core technology for multiplexed molecular analysis of cellular and tissue specimens. However, conventional solution-based staining is labor-intensive and time-consuming and requires considerable expertise to yield optimal results, which creates difficulties for employing this technology in resource-limited settings. Here, we report a new immunostaining method based on hydrogel stamping, which is simple, fast, easy to use, and reproducible. We showed that a hydrophilic hydrogel stamp could effectively transfer fluorescent antibodies to targets and withdraw an excess solution when the reaction is completed, obviating the need for extra washing. This unique property allows for quality immunostaining in 5 min for cells using one-eighth of antibody consumption compared to the conventional solution-based method. Furthermore, we implemented fluorescence quenching and immunocycling with hydrogel staining for multiplexed analysis of 9 protein markers at a single cell level. Finally, we applied the immunocycling method to human breast cancer tissue samples and showed quality immunostaining over a large area (∼2 cm2) in 30 min for molecular subtyping of breast cancer. The hydrogel immunostaining could open new opportunities for rapid, automated, and multiplexed profiling in compact point-of-care systems for molecular cancer diagnosis.

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“…Although not impressive from a whole-organ imaging perspective, this method might find its place in SRM, as 3 h of c-PRESTO resulted in a 120-µm-deep penetration of labels (a depth that was achievable after 2 days in samples subjected to simple diffusion). A prominent upgrade in the category of pressure-assisted immunolabeling was recently presented by Fiorelli et al [97], who developed a simple device in which N2 is pumped until 225 kPa is reached in the system, resulting in a fast, uniform labeling across multiple tested tissues and antibodies. More details regarding the construction of the device can be found both in a preprint and their patent description [98].…”

Section: Insufficient and Heterogeneous Molecular Probe Labellingmentioning

confidence: 99%

“…A prominent upgrade in the category of pressure-assisted immunolabeling was recently presented by Fiorelli et al [97], who developed a simple device in which N2 is pumped until 225 kPa is reached in the system, resulting in a fast, uniform labeling across multiple tested tissues and antibodies. More details regarding the construction of the device can be found both in a preprint and their patent description [98]. The first of the tissue-transforming TOC techniques (CLARITY) was developed by the Deisseroth group, in which an acrylamide-bisacrylamide solution created a protein and nucleic acid entrapping mesh [17].…”

Section: Insufficient and Heterogeneous Molecular Probe Labellingmentioning

confidence: 99%

Can Developments in Tissue Optical Clearing Aid Super-Resolution Microscopy Imaging?

Matryba

Łukasiewicz

Pawłowska

et al. 2021

IJMS

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The rapid development of super-resolution microscopy (SRM) techniques opens new avenues to examine cell and tissue details at a nanometer scale. Due to compatibility with specific labelling approaches, in vivo imaging and the relative ease of sample preparation, SRM appears to be a valuable alternative to laborious electron microscopy techniques. SRM, however, is not free from drawbacks, with the rapid quenching of the fluorescence signal, sensitivity to spherical aberrations and light scattering that typically limits imaging depth up to few micrometers being the most pronounced ones. Recently presented and robustly optimized sets of tissue optical clearing (TOC) techniques turn biological specimens transparent, which greatly increases the tissue thickness that is available for imaging without loss of resolution. Hence, SRM and TOC are naturally synergistic techniques, and a proper combination of these might promptly reveal the three-dimensional structure of entire organs with nanometer resolution. As such, an effort to introduce large-scale volumetric SRM has already started; in this review, we discuss TOC approaches that might be favorable during the preparation of SRM samples. Thus, special emphasis is put on TOC methods that enhance the preservation of fluorescence intensity, offer the homogenous distribution of molecular probes, and vastly decrease spherical aberrations. Finally, we review examples of studies in which both SRM and TOC were successfully applied to study biological systems.

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Enhanced tissue penetration of antibodies through pressurized immunohistochemistry

Cited by 7 publications

References 43 publications

Visualization and analysis of whole depot adipose tissue neural innervation

Visualization and analysis of whole depot adipose tissue neural innervation

Hydrogel Stamping for Rapid, Multiplexed, Point-of-Care Immunostaining of Cells and Tissues

Can Developments in Tissue Optical Clearing Aid Super-Resolution Microscopy Imaging?

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