Format

Send to

Choose Destination
J Neurosci Methods. 2019 Jan 1;311:235-238. doi: 10.1016/j.jneumeth.2018.10.040. Epub 2018 Nov 1.

Reciprocal nerve staining (RNS) for the concurrent detection of choline acetyltransferase and myelin basic protein on paraffin-embedded sections.

Author information

1
New Jersey Center for Biomaterials, Rutgers- The State University of New Jersey, Piscataway, New Jersey, United States.
2
Research Pathology Services, Rutgers -The State University of New Jersey, Piscataway, New Jersey, United States.

Abstract

BACKGROUND:

Objective of our work was to develop a sequential double nonfluorescent immunostaining method which allows the selective identification of myelinated motor fibers in paraffin-embedded samples of peripheral nerves. Motor recovery after a nerve gap-lesion repaired by artificial nerve-guides ("conduits") is often less complete and slower than sensory recovery. The mechanism for this is not fully understood.

NEW METHOD:

Incubation in sheep polyclonal choline acetyltransferase antibody (Abcam 18,736) at dilution of 1:150 was followed by incubation in mouse monoclonal anti-myelin basic protein antibody (Abcam 62,631) at a dilution of 1:5000. Counterstaining was performed with hematoxylin QS (Vector Labs H-3404).

RESULTS:

Immunostaining of choline acetyltransferase and myelin basic protein can be combined together and results show a good contrast between the light brown of the choline acetyltransferase reaction product and the green of myelin basic protein reaction product. Cell nuclei are stained blue. This new protocol retains the advantages of paraffin embedded sections such as (i) having a relatively simple methodology, (ii) years-long storage life, and (iii) easy sharing among laboratories. Comparison with existing method. This specific combinatorial protocol has never been used before on paraffin embedded sections. It has been named "reciprocal nerve staining" (RNS).

CONCLUSIONS:

Routine combination of choline acetyltransferase and myelin basic protein immunostaining provides a highly specific, highly contrasted paraffin-embedded sections where optical differentiation of myelinated motor fibers is easy and straightforward. This method will likely simplify and speed-up the routine histological study of nerve regeneration and will contribute a better identification of the nerve motor component.

KEYWORDS:

Biomaterials; ChAT; Conduit; Gap-lesion; Immunostaining; MBP; Nerve; Paraffin

PMID:
30391262
PMCID:
PMC6319371
[Available on 2020-01-01]
DOI:
10.1016/j.jneumeth.2018.10.040

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center