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. 2025 Jan;48(1):100157.
doi: 10.1016/j.mocell.2024.100157. Epub 2024 Nov 19.

Brief guide to immunostaining

Gyutae Park  1 , Sieun S Kim  1 , Jiwon Shim  2 , Seung-Jae V Lee  3
Affiliations

Brief guide to immunostaining

Gyutae Park et al. Mol Cells. 2025 Jan.

Abstract

Immunostaining is an essential biological technique that determines the localization and level of target antigen molecules using antibodies within cells or tissues. Here, we present a brief guide to immunostaining, including the principles, methods, and different types of immunostaining. This manuscript will also provide common challenges and optimization strategies. This work will be useful for researchers with basic knowledge in immunostaining.

Keywords: Immunocytochemistry; Immunofluorescence; Immunohistochemistry.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.The author is an Editor-in-Chief for Molecules and Cells and was not involved in the editorial review or the decision to publish this article.

Figures

Fig. 1
Fig. 1
Typical workflow of immunostaining. Immunostaining is initiated with cell or tissue preparation, in which specific cell or tissue samples are fixed to preserve their structures. The samples are then incubated with a blocking buffer to prevent nonspecific binding between antigen and antibody. Subsequently, the samples are incubated with a primary antibody that specifically binds to the target antigen, followed by incubation with a secondary antibody linked to a fluorescent dye or an enzyme. After washing to remove excess antibodies, the sample is mounted on a slide glass using a mounting medium. Lastly, the antigen-antibody complexes are visualized using proper microscopy.
See this image and copyright information in PMC

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