Protocol to detect smooth muscle actin-alpha and measure oxidative damage in neonatal mouse intestine

Shing Hu; Carolyn S Sevier; Natasza A Kurpios

doi:10.1016/j.xpro.2022.101524

Protocol to detect smooth muscle actin-alpha and measure oxidative damage in neonatal mouse intestine

STAR Protoc. 2022 Sep 16;3(3):101524. doi: 10.1016/j.xpro.2022.101524. Epub 2022 Jul 9.

Authors

Shing Hu¹, Carolyn S Sevier², Natasza A Kurpios³

Affiliations

¹ Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA. Electronic address: sph58@cornell.edu.
² Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA. Electronic address: carolyn.sevier@cornell.edu.
³ Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA. Electronic address: natasza.kurpios@cornell.edu.

Abstract

This protocol describes how to characterize α-Smooth muscle actin (αSMA) spatiotemporal expression during mouse small intestinal development. Specific tissue fixation preserves αSMA arrangement in low αSMA expressing cells that are conventionally undetectable under αSMA immunofluorescent stain due to inappropriate fixative-caused artificial actin depolymerization. Parallel analysis of αSMA carbonylation allows estimation of oxidative damage in gut muscular lineage. This approach improves the molecular specificity offered by commercialized kits that estimate total protein carbonyl level in cell lysates without protein specificity. For complete details on the use and execution of this protocol, please refer to Hu et al. (2021).

Keywords: Cell Biology; Developmental biology; Molecular Biology.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Actins* / metabolism
Animals
Animals, Newborn
Fixatives / metabolism
Intestines
Mice
Muscle, Smooth* / metabolism
Oxidative Stress

Substances

Actins
Fixatives

Abstract

Publication types

MeSH terms

Substances

Grants and funding