Reviewer
Fanny Ehret
  • Post-Doc, Institut of Anatomy, Medical Faculty of TU Dresden
Research fields
  • Neuroscience
Quantitative Analysis of Gene Expression in RNAscope-processed Brain Tissue
Authors:  Maria E. Secci, Tanner Reed, Virginia Quinlan, Nicholas W. Gilpin and Elizabeth M. Avegno, date: 01/05/2023, view: 1411, Q&A: 0

Molecular characterization of different cell types in rodent brains is a widely used and important approach in neuroscience. Fluorescent detection of transcripts using RNAscope (ACDBio) has quickly became a standard in situ hybridization (ISH) approach. Its sensitivity and specificity allow for the simultaneous detection of between three and forty-eight low abundance mRNAs in single cells (i.e., multiplexing or hiplexing), and, in contrast to other ISH techniques, it is performed in a shorter amount of time. Manual quantification of transcripts is a laborious and time-consuming task even for small portions of a larger tissue section. Herein, we present a protocol for creating high-quality images for quantification of RNAscope-labeled neurons in the rat brain. This protocol uses custom-made scripts within the open-source software QuPath to create an automated workflow for the careful optimization and validation of cell detection parameters. Moreover, we describe a method to derive mRNA signal thresholds using negative controls. This protocol and automated workflow may help scientists to reliably and reproducibly prepare and analyze rodent brain tissue for cell type characterization using RNAscope.


Graphical abstract


Mechanical Fractionation of Cultured Neuronal Cells into Cell Body and Neurite Fractions
Authors:  Ankita Arora, Raeann Goering, Hei-Yong G. Lo and J. Matthew Taliaferro, date: 06/05/2021, view: 4019, Q&A: 0

Many cells contain spatially defined subcellular regions that perform specialized tasks enabled by localized proteins. The subcellular distribution of these localized proteins is often facilitated by the subcellular localization of the RNA molecules that encode them. A key question in the study of this process of RNA localization is the characterization of the transcripts present at a given subcellular location. Historically, experiments aimed at answering this question have centered upon microscopy-based techniques that target one or a few transcripts at a time. However, more recently, the advent of high-throughput RNA sequencing has allowed the transcriptome-wide profiling of the RNA content of subcellular fractions. Here, we present a protocol for the isolation of cell body and neurite fractions from neuronal cells using mechanical fractionation and characterization of their RNA content.


Graphic abstract:



Fractionation of neuronal cells and analysis of subcellular RNA contents


Delayed Alternation Task for the Study of Spatial Working and Long Term Memory in Rats
Authors:  Megi Hoxha and Marta Sabariego, date: 03/05/2020, view: 4488, Q&A: 0
Memory systems can hold previously presented information for several seconds, bridging gaps between discontinuous events. It has been previously demonstrated that the hippocampus and the medial entorhinal cortex (mEC) are necessary for memory retention over delay intervals in alternation tasks. Here we describe the delayed alternation task, a spatial working memory (WM) task in which animals need to alternate between left and right sides of a figure-8 maze on a trial-by-trial basis to receive a reward. On each trial of this task, the rat has to remember the last episode and turn in the opposite direction compared to the previous trial. We manipulated the WM load by introducing delays of various lengths (10 s and 60 s) between trials. While other alternation task protocols use short delay intervals between trials, our protocol introduces a longer delay condition that requires animals to use long-term memory resources that are not necessarily supported by sequential neuronal firing patterns (i.e., time cells) as are seen with shorter delay intervals.
A Simple and Efficient Method for Concomitant Isolation and Culture of Enriched Astroglial and Microglial Cells from the Rat Spinal Cord
Authors:  Pooja Shree Mishra and Trichur R Raju, date: 01/20/2020, view: 4062, Q&A: 0
Investigations into glial biology have contributed substantially in understanding the physiology and pathology of the nervous system. However, intricacies of the neuron-glial and glial-glial interactions in vivo present significant challenges while delineating the individual cell-type contributions, thus making the in vitro techniques exceedingly relevant to study glial biology. However, obtaining optimal yield along with high purity has been challenging for microglial cultures. Here we present a simple protocol to establish enriched astroglial as well as microglial cultures from the neonatal rat spinal cord. This method results in highly enriched astroglial and microglial cultures with maximal yield.
Preparation of Crude Synaptosomal Fractions from Mouse Brains and Spinal Cords
Author:  Oliver Wirths, date: 08/05/2017, view: 13845, Q&A: 0
The current protocol describes the preparation of crude synaptosomal fractions from mouse brain or spinal cord samples. In detail, a sequential protocol yielding crude synaptosomal and light membrane fractions is provided. This fast and easy method might be sufficient to assess the amount of synaptic proteins in down-steam applications like Western-blot or ELISA in e.g., mouse models of Alzheimer’s disease or other neurodegenerative conditions.
Extraction of Soluble and Insoluble Protein Fractions from Mouse Brains and Spinal Cords
Author:  Oliver Wirths, date: 08/05/2017, view: 27283, Q&A: 3
The current protocol details the preparation of soluble and insoluble protein lysates from mouse brain or spinal cord samples. In detail, tissue homogenization and sequential protein extraction are described. This procedure yields soluble and insoluble protein extracts that can be further processed in down-stream applications like ELISA or Western blotting.
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