"Systematic Review of Immunohistochemistry Protocols" by Arpitha Parthasarathy

Researcher ORCID Identifier

0000-0001-9832-6575

Graduation Year

2025

Document Type

Open Access Senior Thesis

Degree Name

Bachelor of Science

Department

Biology

Reader 1

Jenna Monroy

Reader 2

Bethany Caulkins

Terms of Use & License Information

Terms of Use for work posted in Scholarship@Claremont.

Rights Information

@ 2025 Arpitha Parthasarathy

Abstract

Immunohistochemistry (IHC) is an essential technique in biological research that allows for the visualization and localization of proteins within tissue samples. This systematic review analyzes the most effective, efficient and reproducible protocols for IHC. It focuses on three critical steps: freezing, cryosectioning, and staining of skeletal muscle tissues in mice models. A total of 41, 44, and 48 articles on freezing, cryosectioning, and staining techniques, respectively, were analyzed, from reputable sources in biology like PubMed and Web of Science. Results showed the following. For freezing, the use of isopentane combined with liquid nitrogen appeared to be the best method, minimizing ice crystal formation and preserving tissue integrity. Cryosectioning results highlighted the importance of maintaining an ideal section thickness of 7-8 microns and using temperatures between -20°C and -25°C to ensure structural preservation and prevent tissue damage. Staining protocols revealed that primary antibodies such as SC-71 and BF-F3 were highly effective for visualizing myosin heavy chains, while Alexa Fluor 488 stood out as the optimal secondary antibody due to its high signal intensity and photostability.

By synthesizing these findings, this review provides a comprehensive guide for researchers aiming to optimize IHC procedures for skeletal muscle studies where there has been no general consensus in the field. The insights gained from this analysis contribute to improving the reliability and reproducibility of IHC experiments, allowing for a deeper understanding of muscle function, disease mechanisms, and adaptive processes.

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