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.
Keywords: AF, Alexa Fluor; Antigen retrieval; BSA, bovine serum albumin; Brain; Citrate buffer; DAPI, 4′, 6-diamidino-2-phenylindole; HRP, horseradish peroxidase; IF, immunofluorescence; IHC, immunohistochemistry; Immunofluorescence; NDS, normal donkey serum; Neuroscience; PB, phosphate buffer; PBS, phosphate-buffered saline; Phosphate buffer; RT, room temperature; SDS, sodium dodecyl sulfate; SNR, signal-to-noise ratio; Signal intensity; Signal-to-noise ratio; Sodium dodecyl sulfate; TB, tris buffer; TBS, tris-buffered saline; TH, tyrosine Hydroxylase.