Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

CARGO: A Cytometry Analysis framework via Regularized Graph Optimal-transport.

PLoS computational biology·2026
Same author

Revealing biological mechanisms of skin pigment recovery during treatment in vitiligo patients: A proteomic analysis.

The Journal of investigative dermatology·2026
Same author

Exploring novel skin biomarkers related to pruritus in dialysis patients.

International urology and nephrology·2026
Same author

Automated Computational Flow Cytometry Correlates Decreasing Neutrophil-to-Lymphocyte Ratio to Improved Survival in NSCLC After Immune Checkpoint Blockade.

Cancer immunology research·2026
Same author

REACtiVe-2: phase I evaluation of dendritic cell vaccination and agonistic CD40 therapy following (m)FOLFIRINOX in metastatic pancreatic cancer.

Nature communications·2025
Same author

[Vitiligo].

Nederlands tijdschrift voor geneeskunde·2025

Related Experiment Video

Updated: Nov 10, 2025

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens
10:49

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Published on: January 21, 2019

21.1K

Improvement of Opal Multiplex Immunofluorescence Workflow for Human Tissue Sections.

Marcella Willemsen1,2,3,4, Gabrielle Krebbers1,2,3,4, Marcel W Bekkenk1,2,3,4

  • 1Department of Dermatology, Amsterdam University Medical Centers, location AMC, University of Amsterdam, Amsterdam, The Netherlands.

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society
|April 2, 2021
PubMed
Summary

A new method using beta-mercaptoethanol buffer effectively removes antibodies in Opal multiplexing, preserving tissue morphology and improving biomarker detection compared to harsh heating methods.

Keywords:
fluorescent antibody techniqueimmunohistochemistryskin

More Related Videos

Author Spotlight: Unlocking Insights into the Immune Cell Landscape of Tumors
06:32

Author Spotlight: Unlocking Insights into the Immune Cell Landscape of Tumors

Published on: August 18, 2023

2.5K
Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma
07:52

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Published on: January 9, 2019

20.1K

Related Experiment Videos

Last Updated: Nov 10, 2025

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens
10:49

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Published on: January 21, 2019

21.1K
Author Spotlight: Unlocking Insights into the Immune Cell Landscape of Tumors
06:32

Author Spotlight: Unlocking Insights into the Immune Cell Landscape of Tumors

Published on: August 18, 2023

2.5K
Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma
07:52

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Published on: January 9, 2019

20.1K

Area of Science:

  • Biotechnology
  • Histology
  • Immunohistochemistry

Background:

  • The Opal multiplex technique enables simultaneous detection of multiple biomarkers on a single tissue section.
  • Current protocols involve iterative antibody removal via heating, which can cause significant tissue damage, particularly in delicate or low-density tissues.
  • There is a need for milder antibody stripping methods to preserve tissue integrity during multiplexed biomarker analysis.

Purpose of the Study:

  • To evaluate the efficacy of beta-mercaptoethanol-based buffer as a gentler alternative to heat-mediated antibody stripping in the Opal multiplex technique.
  • To assess the impact of this alternative stripping method on tissue morphology and biomarker staining intensity.
  • To determine if staining intensity can be recovered following beta-mercaptoethanol treatment.

Main Methods:

  • The Opal multiplex technique was applied to various tissue types, including skin and low-cell-density tissues.
  • Antibody stripping was performed using either traditional heating cycles or a beta-mercaptoethanol-containing buffer.
  • Tissue morphology and biomarker staining intensity were analyzed after repetitive stripping cycles.
  • Primary antibody concentrations were adjusted to assess recovery of staining intensity.

Main Results:

  • Repetitive heating cycles caused considerable morphological damage to various tissue types.
  • Using beta-mercaptoethanol buffer for antibody removal preserved tissue morphology effectively.
  • While some target epitopes showed reduced staining intensity after multiple buffer treatments, this was fully reversible by increasing primary antibody concentrations.
  • The beta-mercaptoethanol method significantly enhanced the quality of Opal multiplexing, allowing visualization of more multicolored cells within intact tissue.

Conclusions:

  • Beta-mercaptoethanol-based buffer is a superior alternative to heat for antibody stripping in the Opal multiplex technique, minimizing tissue damage.
  • This optimized protocol preserves tissue morphology while enabling robust multiplex biomarker detection.
  • The findings support the broader application of Opal multiplexing across diverse tissue types by improving protocol robustness and data quality.