Application of NeuroTrace staining in the fresh frozen brain samples to laser microdissection combined with quantitative RT-PCR analysis

Abstract

BackgroundThe heterogeneity of the brain requires appropriate molecular biological approaches to account for its morphological complexity. Laser-assisted microdissection followed by transcript profiling by quantitative determination has been reported to be an optimal methodology. Nevertheless, not all brain regions can be identified easily without staining, restricting the accuracy and efficiency in sampling. The aim of the present study was to validate whether fixation and staining treatments are suitable for quantitative transcript expression analysis in laser microdissection (LMD) samples. Quantitative RT-PCR was used to determine the absolute transcript expression levels and profiles of samples obtained from the hippocampal dentate gyrus from fresh frozen mice brain sections that had been fixed with ethanol and stained with NeuroTrace. The results were compared with those obtained from unfixed and unstained samples.ResultsWe found that the quantitative relationship of transcript expression levels between various housekeeping genes and immediate early genes was preserved, although the preparation compromised the yield of the transcripts. In addition, histological and molecular integrities of the fixed and stained specimens were preserved for at least a week at room temperature. Based on the lobe specific profiles of transcripts in the anterior and posterior lobes of the pituitary, we confirmed that no cross-contamination on transcription expressions occurred as a result of the fixation and staining.ConclusionsWe have provided detailed information of the procedures on ethanol fixation followed by NeuroTrace staining on the absolute quantitative RT-PCR analysis using microdissected fresh frozen mouse brain tissues. The present study demonstrated that quantitative transcript expression analysis can be conducted reliably on stained tissues. This method is suitable for applications in basic and clinical studies on particular transcript expressions in various regions of the brain.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1222-9) contains supplementary material, which is available to authorized users.

β-amyloid monomer scavenging by an anticalin protein prevents neuronal hyperactivity in mouse models of Alzheimer’s Disease

“…After 18 h, the mice were perfused intracardially with 4% PFA and the brains were post-fixed overnight 71 . The next day, coronal sections were prepared and NeuroTrace deep red counterstaining was performed 72 . Slices were mounted in a confocal microscope (Olympus) and images were taken using a 4X air immersion objective and stitched together.…”

Mapping Molecular Datasets Back to the Brain Regions They are Extracted from: Remembering the Native Countries of Hypothalamic Expatriates and Refugees

“…Importantly, investigators have performed LCM on the Nissl-stained tissue itself [43, 208, 350, 390], but in principle, one can also use adjacent sections stained for Nissl substance to help delineate regions of interest on unstained companion sections sampled by LCM, as has been done for human tissue samples collected post mortem for the PVH and SO [451]. In addition to using Nissl staining as a tool to help delineate LCM-captured tissue sample boundary conditions, other stains and labeling strategies have also been used in conjunction with LCM, including FluoroJade for delimiting tissue pathology [43], Cy3-conjugated secondary antibody to identify antibody-labeled peptidergic neurons [302–304], immunoperoxidase-based detection of peptidergic neurons [47], NeuroTrace staining for visualizing fluorescent Nissl-like profiles [31], and in situ hybridization in human post mortem tissue [33]. Finally, it is worth noting that although LCM procedures themselves do not appear to result in significant losses of protein as compared to manually dissected samples of comparably located regions, Nissl staining itself can be detrimental to the full retention of some proteins for subsequent proteomic analyses [290], and the use of Nissl stains such as neutral red, cresyl violet, or NeuroTrace reportedly contributes to lower yields of transcripts from LCM-captured brain tissue [31, 213].…”

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“…NeuroTraceNeuroTrace fluorescent Nissl stain is selective for the Nissl substance present in neurons[101]. Nissl substance is composed of ribosomal RNA associated with the rough endoplasmic reticulum in neuronal perikarya and dendrites.…”