Improved method for combination of immunocytochemistry and Nissl staining

Abstract

Nissl staining is a widely used method to study morphology and pathology of neural tissue. After standard immunocytochemistry, the Nissl staining labels only the nucleus of neurons and the characteristic staining of the neuronal perikarya is absent or very weak. We hypothesized that the RNA degradation during the immunocytochemical treatment results in the loss of cytoplasmic staining with Nissl-dyes. To test this hypothesis, we used RNAse-free conditions for all steps of immunostaining. To further prevent the RNA-degradation by RNAse contaminations, the RNAse inhibitor heparin was added to all antibody-containing solutions. The efficiency of Nissl staining after standard and RNAse-free double-labeling immunocytochemistry was compared using antibodies against c-Fos and neuropeptide Y (NPY) on tissues of rats refed after 3 days of fasting. After standard immunocytochemistry, the Nissl-staining labeled the nuclei of neurons and only very faintly the cytoplasm of these cells. The RNAse-free treatment did not alter the distribution of immunoreaction signal, but preserved the staining of neuronal perikarya by the Nissl-dyes. In conclusion, the RNAse-free conditions during immunocytochemistry allow the labeling of neuronal perikarya by Nissl-dyes. The described method facilitates the mapping of immunocytochemical signals and makes possible the light microscopic examination of the innervation of neurons identified by their nuclear protein content.

Figure 1

Combination of double-labeling immunocytochemistry for NPY (brown) and c-Fos with Nissl-staining (violet) using standard (A, C, E) and RNAse free (B, D, F) immunocytochemical techniques on sections containing the hypothalamic paraventricular nucleus (PVN) of rats refed after three day fasting. Low and medium power micrographs illustrate the distribution of NPY- and c-Fos-IR elements in the PVN (A, B) and in the ventral parvocellular part of the PVN (C–F), respectively. The distribution of the immunoreaction signals is identical on section immunostained with standard (A, C) or RNAse free (B, D) method, however, the Nissl staining resulted in only nuclear or pale cytoplasmic labeling after the standard immunocytochemistry (A, C) while strong cytoplasmic Nissl-staining is observed after RNAse free immunocytochemistry (B, D). High power micrographs (E, F) illustrate the the relationship of NPY-IR varicosities and the c-Fos-IR neurons in the ventral parvocellular subdivision of the PVN. Although, NPY-IR varicosities surround the c-Fos-IR neurons of both images (E, F), the cytoplasmic border of c-Fos-IR neurons and the juxtaposition of NPY-IR varicosities (arrows) and the c-Fos-IR neurons can be recognized only on the preparations stained with RNAse free immunocytochemistry (F). Scale bar on B = 100 μm corresponds to A and B, scale bar on D = 100 μm corresponds to C and D, scale bar on F = 20 μm corresponds to E and F.