American Society for Peripheral Nerve

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Reciprocal Nerve Staining (RNS) Allows the Concurrent Detection of Myelin Basic Protein and Choline Acetyltransferase on Paraffin-embedded Sections
Antonio Merolli, MD FBSE1; Pedro Louro, MBA1; Cristiana Carvalho, PhD2; Joachim Kohn, PhD FBSE1
1Rutgers - The State University of New Jersey, Piscataway, NJ, 2University of Minho, Guimaraes, Portugal

Introduction. Myelin Basic Protein (MBP) immunostaining is a well established methodology to differentiate between myelinated and not myelinated fibers. Choline acetyltransferase immunostaining can differentiate between motor and non-motor (largely sensory) fibers. These methodologies, largely employed in cryosection and fluorescence microscopy, have been recently individually applied to paraffin-embedded sections. Traditional paraffin embedded sections present the advantage to have a relatively simple methodology and allow decades long storage life. They can be easily shared among laboratories worldwide. In the study of nerve regeneration, a clear-cut differentiation between motor and sensory fibers is critical; at the same time, myelinated fibers must be clearly identified. We developed a histological protocol where both MBP and ChAT are stained together with sufficient contrast on paraffin-embedded sections. The technique has been called reciprocal nerve staining (RNS).

Materials and Methods. The sections were deparaffinized, hydrated with alcohol, and subjected to heat-induced epitope retrieval with citrate buffer, pH 6.0 for 20 minutes at 98 degrees using. The sections were blocked with 10% normal donkey serum for 30 minutes followed by 48-hour incubation of sheep polyclonal to choline acetyltransferase antibody (Abcam 18736) at dilution of 1:150. The secondary antibody donkey anti-sheep polymer was used followed by 5 minutes of DAB chromogen substrate (Vector Labs SK-4105). After completion of 1st primary antibody (ChAT), slides were again subjected to heat-induced epitope retrieval with citrate buffer, pH 6.0 for 20 minutes at 98 degrees. The sections were blocked with 10% normal horse serum for 30 minutes followed by 1 hour incubation of mouse monoclonal anti-myelin basic protein antibody (Abcam 62631) at a dilution of 1:5,000. The secondary antibody ImmPRESS VR anti-mouse IgG HRP Polymer Detection Kit was used (Vector Labs) followed by 30 minutes of Vina Green chromogen substrate (Biocare Medical). Counterstaining was performed with hematoxylin QS (Vector Labs H-3404).

Results. Figure 1 shows the contrast between the green of myelin basic protein and the orange-yellow of the choline acetyltransferase. Nuclei are identified thanks to the counterstaining performed with hematoxylin.



Conclusions. The routine combination of choline acetyltransferase (ChAT) and myelin basic protein (MBP) immuno-histochemistry may prodive highly specific, highly contrasted paraffin-embedded sections and has not yet been reported in the literature, to the best of our knowledge. In this way, we can easily differentiate between myelinated and nonmyelinated fibers. At the same time, differentiation between myelinated motor fibers and myelinated sensory fibers becomes intuitive as well.


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