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

Samer Salameh,Dominique Nouel,Cecilia Flores,Daniel Hoops

doi:10.1016/j.mex.2017.12.001

Samer Salameh, Dominique Nouel + Show 2 more

Open Access

https://doi.org/10.1016/j.mex.2017.12.001

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Journal: MethodsX	Publication Date: Dec 13, 2017
Citations: 4	License type: cc-by

Affiliation: Douglas Mental Health University Institute

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.

Highlights

The key protocol modifications are as follows: Use phosphate buffer (PB) as the blocking solution solvent
This protocol consists of steps 6–13 as described above except that the blocking solution is made with phosphate-buffered saline (PBS), not phosphate buffer (PB), and all rinses are performed using PBS
We found that heating sections prior to blocking resulted in Netrin-1 staining; the Netrin-1 signal was weaker than with sodium dodecyl sulfate (SDS)

Summary

Introduction

The key protocol modifications are as follows: Use phosphate buffer (PB) as the blocking solution solvent. Netrin-1 fluorescence immunohistochemistry protocol (Novus brand antibody) 4. Place sections in a 1% sodium dodecyl sulfate (SDS) solution for 5 min on an orbital shaker at room temperature (RT).

Results

Conclusion