An optimized immunohistochemistry protocol for detecting the guidance cue Netrin-1 in neural tissue.

TitleAn optimized immunohistochemistry protocol for detecting the guidance cue Netrin-1 in neural tissue.
Publication TypeJournal Article
Year of Publication2018
AuthorsSalameh S, Nouel D, Flores C, Hoops D
JournalMethodsX
Volume5
Pagination1-7
Date Published2018
ISSN2215-0161
Abstract

Netrin-1, an axon guidance protein, is difficult to detect using immunohistochemistry. We performed a multi-step, blinded, and controlled protocol optimization procedure to establish an efficient and effective fluorescent immunohistochemistry protocol for characterizing Netrin-1 expression. Coronal mouse brain sections were used to test numerous antigen retrieval methods and combinations thereof in order to optimize the stain quality of a commercially available Netrin-1 antibody. Stain quality was evaluated by experienced neuroanatomists for two criteria: signal intensity and signal-to-noise ratio. After five rounds of testing protocol variants, we established a modified immunohistochemistry protocol that produced a Netrin-1 signal with good signal intensity and a high signal-to-noise ratio. The key protocol modifications are as follows: •Use phosphate buffer (PB) as the blocking solution solvent.•Use 1% sodium dodecyl sulfate (SDS) treatment for antigen retrieval. The original protocol was optimized for use with the Netrin-1 antibody produced by Novus Biologicals. However, we subsequently further modified the protocol to work with the antibody produced by Abcam. The Abcam protocol uses PBS as the blocking solution solvent and adds a citrate buffer antigen retrieval step.

DOI10.1016/j.mex.2017.12.001
Alternate JournalMethodsX
PubMed ID29276694
PubMed Central IDPMC5737949
Grant ListR01 DA037911 / DA / NIDA NIH HHS / United States

  • Douglas Hospital
  • Dobell Pavillion
  • Brain imaging centre