Detecting Neural Pathways

Making Headway in Neural Pathways

Mapping the sensitivity and variability of the brain is crucial for interpreting findings and driving new discoveries in neuroscience research. With innovative technologies in modulation, screening and visualization, scientists can now further their understanding of the brain. This growth of knowledge and information of the nervous system allows you to better treat neurological diseases and ultimately improve the human condition.

Discovering neural pathways involves three processes:

modulatescreencharacterize

Use this guide to overcome challenges and learn more about your neural pathway.

 

MODULATE > Modify the system

With the use of gene silencing and bioactive small molecules neuroscientists have a high degree of control over their neural pathways. Precise modulation of neural function enables the correlation of cellular response to pathway biology and ultimately, mechanisms of diseases, making a big impact in neuroscience research.

CHALLENGE

There is a need for targeted modulation so scientists can understand the role of pathways in and between cells of the nervous system.1

SOLUTION

Gene Silencing Our large portfolio of RNA interference (RNAi) tools (siRNA, esiRNA, shRNA, miRNA) allow researchers to investigate gene function and silence selected genes in order to study phenotypic responses and the role they may play in the field of neuroscience. The choice of method depends on a variety of factors, including susceptibility to transfection and desired knockdown duration. The study and manipulation of RNAi is a valued experimental tool for biomedical research that can, for example, lead to discovering new therapeutic targets for neurological diseases or identifying nerve sheath tumor driver genes.1

1EBioMedicine. 2016 Jul; 9: 110-119.

Learn more at sigmaaldrich.com/RNAi


SOLUTION

Bioactive Small Molecules
Bioactive small molecules (BSM) provide an essential means for controlling cellular process in order to understand the underlying biology of neural pathways. From preventing a process from occurring, stopping cell division or stimulating a different process, BSMs can control the activity of protein targets and facilitate the study of mechanisms that underlie disease. The discoveries made with BSMs allow scientists to work on the next generation neurological treatments (e.g. stem cell and personalized medicine). Count on our high quality, well-cited agonists, antagonists and modulators for targeted manipulation of neuroscience pathways. Highlighted neural specific BSMs are shown below.

Bioactive Small Molecules

Product
No.
Description Biochem/physio Actions
D5942 DAPT ≥98%
(HPLC), solid
DAPT is a γ-secretase inhibitor and indirectly an inhibitor of Notch, a γ-secretase substrate. Other γ-secretase substrates include LDL receptor-related protein, E-cadherin and ErbB-4. As an inhibitor of γ-secretase, DAPT may be useful in the study of β-amyloid (Aβ) formation. DAPT has been shown to inhibit Notch signaling in studies of autoimmune and lymphoproliferative diseases, such as ALPS and systemic lupus erythematosus (SLE), as well as in cancer cell growth, angiogenesis, and differentiation of human induced pluripotent stem cells (hIPSC).
M9292 Memantine
hydrochloride
≥98% (GC)
Low-affinity NMDA glutamate receptor antagonist; stimulates dopamine release.
PZ0010
Pregabalin ≥97%
(NMR)
Pregabalin is a lipophilic GABA analog/ligand at α2δ subunit of voltage-dependent Ca2+ channels. Pregabalin is an anticonvulsant, anxiolytic analgesic used to treat peripheral neuropathic pain and fibromyalgia.
PZ0004 Varenicline tartrate
≥98% (HPLC)
Varenicline tartrate is a partial α4β2 nicotinic receptor agonist and α7 full agonist. Varenicline competitively binds to α4β2 receptors and partially stimulates without creating a full nicotine effect, while simultaneoudly blocking the ability of nicotine to bind to the receptors. Varenicline thus blocks the ability of nicotine to activate α4β2 receptors and stimulate the central nervous mesolimbic dopamine system, believed to be the neuronal mechanism underlying reinforcement and reward experienced upon smoking.
BM0005 Avagacestat ≥98%
(HPLC)
Avagacestat (BMS-708163) is a potent γ-secretase inhibitor selective for amyloid precursor protein (APP) over Notch cleavage. Avagacestat inhibits Aβ40 and Aβ42 production with IC50 values of 0.3 nM and 0.27 nM, respectively, compared to IC50values of 41, 10, 33, and 38 nM respectively for signaling inhibition of the human Notch 1-4 proteins. At higher concentrations BMS-708163 has been found to reverse sensitivity to the EGFR tyrosine kinase inhibitor gefitinib, thought to be by Notch inhibition.
SML1526 Eltoprazine
hydrochloride
≥98% (HPLC)
Eltoprazine hydrochloride is a serotonin 5-HT1A/1B receptor agonist that has been shown to reduce L-DOPA-induced dyskinesias in in experimental models of Parkinson′s Disease and in human patients. It has Ki values for 5-HT1A, 5-HT1B and 5-HT1C receptors of 40 nM and 52 nM, respectively.

TECHNOLOGY HIGHLIGHT

BSM Resource Guide

The complexity of the nervous system is illustrated by the diverse array of receptors, neurotransmitters, transporters and ion channels that are involved in signal transmission. To better understand components involved in neurotransmission, reference our eHandbook for receptor classification and signal transduction information at www.sigma.com/bsm-handbook.

Example of components involved in neurotransmission:

Glutamate
   G Protein, ion channel receptors
GABA
   GABAA, GABAB, GABAC receptors, transporters
Serotonin
   Receptors, synthesis, metabolism
Dopamine
   Receptors, synthesis, metabolism

Discover our comprehensive portfolio of BSMs at www.sigmaaldrich.com/bioactive

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SCREEN > Novel Targets and Pathways

The ability to screen large numbers of DNA/RNA/proteins involved in neurological diseases can result in identifying new cellular pathways and potentially new therapeutics (e.g. specifically inhibit the disease-causing mechanisms of cellular targets). Count on our powerful technologies to screen genes and small molecules to thoroughly examine neural pathways.

CHALLENGE

Within complex neural pathways there is a need to identify novel targets.

SOLUTION

Gene knockout
CRISPR Cas9 nucleases have revolutionized the field of gene editing and highthroughput lentiviral screens continue to hold ever-increasing promise for both basic research and development of future therapies to benefit human health. Explore these genetic screens for neural pathway target discovery.

Sanger library
Genome-wide loss-of-function screening is a powerful approach to discover genes and pathways that underlie biological processes in neuroscience. We have partnered with The Wellcome Sanger Institute to make the first arrayed lentiviral CRISPR knockout library for human and mouse genomes.

  • Whole genome arrayed Sanger libraries for human and mouse with BFP detection

Sanger Lentiviral CRISPR vector schematic

Sanger Lentiviral CRISPR vector schematic (U6-gRNA:PGK-puro-2A-tagBFP)
 

GeCKO libraries

  • Whole genome pooled GeCKO libraries for human and mouse with GFP detection

Custom libraries- arrayed or pooled

  • Custom GeCKO and Sigma pools for focused pooled screening projects
  • Custom Sanger arrayed panels for projects (e.g. 2-2000 genes)

Learn more at sigmaaldrich.com/neuro-screening

 

SOLUTION

RNA interference
RNA interference (RNAi), either synthetic small interfering RNA (siRNA) or the vector-based short hairpin RNA (shRNA), is an important tool to study genes by silencing gene function and examine which processes are affected in neural pathways. RNAi screening is a very powerful tool to map out gene functions, identify redundant genes that perform similar or compensatory functions, as well as validate predicted (hypothetical) gene functions.

MISSION® shRNA library
The most comprehensive and thoroughly validated shRNA collection available, MISSION® shRNA library is an exclusive technology and contains almost 250,000 clones including more than 80,000 validated clones. It combines all of the highly trusted TRC1 content plus an additional 88,000 clones targeting 4,000 new human genes and more than 5,000 new mouse genes.

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TECHNOLOGY HIGHLIGHT

Need tools to create your cell based assay?

Live cell imaging tools
Flow cytometry and antibody based assays are a great way to capture a snap shot of cellular events. However, if you want to track real time changes in cell based assays, chelators and ion probes can be used to track ionic concentrations in fluorescent measurements and provide information on your assay including cell viability, ions, pH, nitric oxide or oxygen. Because slight ionic changes can indicate deviations in neuronal function, count on our fluorescent dyes, probes, and biosensors to build your cell based assay and monitor your area of interest.

PKH/CellVue® Cell Labeling Kits: PKH and CellVue® Fluorescent Cell Linker Kits provide fluorescent membrane labeling of live cells over an extended period of time. Learn more at sigmaaldrich.com/neuro-labeling

LentiBrite™ Fluorescent Biosensors: Pre-packaged high-titer fluorescent lentiviral particles encoding GFP/RFP labeled proteins for topics such as autophagy, apoptosis, and cell structure.

SOLUTION

Chemical libraries
High-throughput analyses, such as chemical libraries, allow neuroscientists to screen against a specific target or neural disease model and test to see if any of the chemicals exhibit the preferred activity. Then those selected chemicals are further validated to advance neurological drug development.

LOPAC®1280 – Library of pharmacologically active compounds
This biologically annotated collection of inhibitors, receptor ligands, pharma-developed tools, and approved drugs impacts most signaling pathways and covers all major drug target classes. With 1,280 bioactive small molecules and wellcharacterized activities our LOPAC®1280 library allows neuroscientists to perform:

  • Assay validation
  • Phenotypic screening / chemical genetics
  • Target characterization

breakdown of LOPAC1280 active compounds


Pharma Developed Compounds

We have partnered with some of the largest pharma companies to allow access to over 800 high quality, validated compounds, including approved therapeutics and drug candidates. Marketed drugs like varenicline to treat nicotine addiction, ziprasidone for psychosis, and the anti-inflammatory celecoxib,as well as many other highly characterized compounds are included in the portfolio. These are available individually for your research and screening needs.

Custom Libraries
Aldrich Market Select is a comprehensive chemical database of building blocks and screening compounds for early stage discovery efforts and custom libraries. It consists of over 8 million chemical products; represented by over 6 million unique chemical structures; from more than 60 chemical suppliers around the world. Regular maintenance assures you can be confident you’re viewing the most recent catalog data. With our custom library services we can assist you in the creation of focused or diverse libraries designed for your specific research needs.

Learn more at sigmaaldrich.com/neuro-bsm

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CHARACTERIZE > Visualize and Detect

Neurons are dynamic entities, sensing and responding to their immediate environment. For this reason, understanding these protein interactions and signaling mechanisms is a focus of neuroscience research and key to understanding normal and disease processes.

CHALLENGE

Acquire visual information on protein interactions in their native state.

SOLUTION

Protein Detection Technology
The chance to discover the inner workings of the brain and its components requires a powerful investigative tool to allow scientists to visualize those protein interactions. With Duolink® proximity ligation assay (PLA) technology, neuroscientists can visualize protein interactions, locations and quantities by amplifying signals corresponding to single protein events. This protein detection technology lets researchers visualize protein functions under true biological conditions without overexpression or genetic manipulation, providing a significant resource in advancing neuroscience.

Learn more at sigmaaldrich.com/neuro-duolink

APPLICATION HIGHLIGHT

Mouse Coronal Tissue Analysis with Duolink® PLA

  • Duolink® PLA was performed on commercially available mouse Caudate Putamen (striatum) frozen floating sections.
  • Brains were fixed overnight in 10% buffered formalin, cryoprotected for 24-36 hours, snapped frozen in O.C.T. medium, and sectioned at a thickness of 30 microns. A polyclonal rabbit antibody targeting the human phosphorylated MAPK pp44/p42 and Duolink® PLA was used.
  • Visualized in a standard epi-fluorescence microscope.
  • All reactions generated robust and distinct Duolink® PLA staining.
Product No. Description
DUO92106 Duolink® In Situ Orange Starter Kit Goat/Rabbit
DUO92104 Duolink® In Situ Orange Starter Kit Mouse/Goat
DUO92102
Duolink® In Situ Orange Starter Kit Mouse/Rabbit
DUO92105 Duolink® In Situ Red Starter Kit Goat/Rabbit
DUO92103 Duolink® In Situ Red Starter Kit Mouse/Goat
DUO92101 Duolink® In Situ Red Starter Kit Mouse/Rabbit

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CHARACTERIZE > Visualize and Detect

CHALLENGE

Detecting changes in neural cell structure and function correlating to development and disease.

SOLUTION

Targeted Antibodies – Application Tested

With advancements in antibody-based biomarker detection reagents, specific and robust primary antibodies, and improved immunodetection techniques, neuroscience is being transformed. We offer neuroscientists the broadest range of trusted antibodies, fully validated in neuro-specific tissues and cell lines for a wide variety of applications (e.g. IHC, WB, ICC), so researchers can better explore and understand the mechanisms of neurological diseases.

Neurodegeneration
neurodegeneration related antibodies The progressive loss of neuronal function can lead to serious debilitating diseases (e.g. Alzheimer's) and cause premature death. This loss of function (neurodegeneration) has been attributed to cell signaling dysfunction, plaque formations, and membrane damage, and programmed cell death.

(image on right) Beta-Amyloid (Cat. No. AB5078P) staining on Alzheimer’s Disease-Brain. Tissue pretreated with Citrate, pH 6.0. Polyclonal Ab

Product No. Description Clonality Species
Origin
Applications
(legend below)
AB5320 Anti-NG2 Chondroitin Sulfate Proteoglycan Antibody Polyclonal Rabbit ELISA, ICC,
IHC, IP, WB
AB5078P Anti-Beta-Amyloid 1-42 Antibody Polyclonal Rabbit ELISA, IHC,
WB
AB5038 Anti-Synuclein α Antibody
Polyclonal Rabbit ICC, IHC,
WB
MAB348 Anti-APP A4 Antibody
Monoclonal-
22C11
Mouse IF, IHC, WB
MAB2166 Anti-Huntingtin Protein Antibody Monoclonal-
1HU-4C8
Mouse ELISA, ICC,
IHC, IP, WB

 

Neuroregeneration
neuroregeneration related antibodies Neuronal development, differentiation, and growth are controlled by a variety of chemical signals and interplay of various transcription factors. Neuronal identity is established by the action of transcriptional programs initiated by the temporal action of transcription factors.


(image on right) Glial cells cultured from rat amygdala. Nuclei stained blue with DAPI
(Cat. No. D9542) and glial processes stained green with Rabbit Anti- GFAP (Cat. No. G9269)

 

Product No. Description Clonality Species
Origin
Applications
(legend below)
AB9610 Anti-Olig-2 Antibody Polyclonal Rabbit ICC, IHC, IP,
WB
ABD69
Anti-Nestin, Human Antibody


Monoclonal-
9H10
Hamster WB, ICC, IHC
AB1554 Anti-Nerve Growth Factor Receptor Antibody, p75 Polyclonal Rabbit ICC, IHC, IP
G9269 Anti-Glial Fibrillary Acidic Protein (GFAP) Antibody Polyclonal Rabbit FC, IHC, ICC
HPA004765 Anti-NGFR antibody Polyclonal Rabbit WB, IHC

 

Cell Structure Antibodies
cell structure antibodies Neuronal cell structure immunostaining is commonly used to visualize morphological changes that occur as a function of a disease state or perturbation and to demonstrate the localization of other biomarkers relative to organelle or cytoskeletal structure.


(image on right) Mouse anti-NeuN (Cat. No. MAB377) and Rabbit anti-Substance P Receptor
(Cat. No. AB5060) staining of normal rat hippocampus.

Product No. Description Clonality Species
Origin
Applications
(legend below)
MAB377 Anti-NeuN Antibody Monoclonal-A60 Rabbit FC, ICC, IF, IHC,
IP, WB
MAB345 Anti-O4 Antibody Monoclonal-81 Mouse ICC, IHC
T8578 Anti-β- Tubulin III Antibody Monoclonal-2G10 Mouse ICC, IHC, IP, WB
MAB3420 Anti-Tau-1 Antibody Monoclonal-PC1C6 Mouse IHC, WB
AB5622 Anti-MAP2 Antibody Polyclonal Rabbit ICC, IHC, WB, IF

 

Cell Function Antibodies
cell function antibodies To monitor and evaluate the ability for neurons to thrive and communicate through neurotransmission is fundamental to this area of research. A wide range of proteins are involved in neuron signaling and regulation of neurotransmitters.


(image on right) anti-Tyrosine Hydroxylase (TH) staining in mouse primary neural cultures using (Cat. No. AB152) shown with an FITC fluorescent secondary (green).

Product No. Description Clonality Species
Origin
Applications
(legend below)
AB152 Anti-Tyrosine Hydroxylase Antibody
Polyclonal Rabbit ELISA, IF,
IHC, IP, WB
AB144P Anti-Choline Acetyltransferase Antibody Polyclonal Goat ICC, IHC,
WB
MAB5406 Anti-GAD67 Antibody Monoclonal-
1G10.2
Mouse IHC, WB
AB1506 Anti-Glutamate Receptor 2 & 3 Antibody Polyclonal Rabbit IHC, ICC, IP,
WB
S5768 Anti-Synaptophysin Antibody Polyclonal Rabbit ELISA, IHC,
WB

 

FC = Flow Cytometry; ICC = Immunocytochemistry; IHC = Immunohistochemistry; IF = Immunofluorescence; IP = Immunoprecipitation; WB = Western Blot

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Prestige Antibodies
When detecting neural pathways, you can rely with confidence on our target-qualified Prestige Antibodies® that are supported with neural-specific images and access to all characterization data.

  • Each antibody is supported by over 700 IHC, IF and Western blot images
  • Developed by the Human Protein Atlas (HPA) Project
  • All data is publically available on the Human Protein Atlas website

The Human Protein Atlas (proteinatlas.org)
displays a collection of over 13 million images of normal and disease tissues, cell lines, and primary cells. The database not only delivers visible proof of antibody performance, but also provides a knowledge base with regard to the structural and temporal expression of proteins in various cells and tissues.


Validation in Human Neuro Tissues and Cell Lines

IHC images from human cerebellum, hippocampus, lateral ventricle wall and cerebral cortex tissues are available for the antibodies, as well as from stainings in the following brain cell lines: D341 Med, SHSY5Y, U-138 MG, U-251 MG, U-87 MG. Malignant glioma tumor samples from up to 12 patients are presented for each antibody in the Cancer Atlas. In addition to IHC images, there are available immunofluorescence (ICC-IF) images from staining in U-251 MG cells for subcellular location information of the proteins.

Immunohistochemical micrographs show dopaminergic system in mouse brain

Sodium-dependent dopamine transporter (SLC6A3, DAT) terminates action of dopamine by its re-uptake into presynaptic terminals. Immunohistochemical micrographs show dopaminergic system in mouse brain using the Anti-DAT antibody (HPA013602). Note strong labelling of dopaminergic cell bodies in the substantia nigra and in terminals in the basal forebrain (insets).

Neuroscience Marker Panel
Our newly developed neuroscience marker panel is designed to recognize the main anatomical and neurochemical cell types in rodent and human nervous system. We have taken great care to be able to offer these markers as tools for mapping the structures and cell types in the central and peripheral nervous system.

  • Selected target proteins are expressed only by a single cell type
  • IHC-validation in rat, mouse and human tissues
  • WB-validation in mouse and human tissue lysates for the majority of the markers
  • Antibodies of different isotypes, allowing for multiplexing experiments

Advance your detection in neural pathways and explore our diverse classification of antibodies in: signaling, neural lineage markers, aging/neurological disorders, and development.

Signaling

Product No. Description Clonality Species
Origin
Applications
(legend below)
HPA001562 Anti-ATF3 antibody Polyclonal Rabbit WB, IF, IHC
HPA013602 Anti- SLC6A3 antibody
Polyclonal Rabbit IHC
AMAB91041 Monoclonal anti-VGLUT1 antibody Cl2754,
Monoclonal
Mouse WB, IHC
AMAB91130 Monoclonal anti-chat antibody Cl3173,
Monoclonal
Mouse IHC

 

Neural Lineage Markers

Product No. Description Clonality Species
Origin
Applications
(legend below)
HPA048982
Anti-CD68 antibody
Polyclonal Rabbit WB,IHC
HPA015768 Anti-S100B antibody
Polyclonal Rabbit WB, IF, IHC
HPA006641 Anti-SCGN antibody Polyclonal Rabbit IHC
HPA056030 Anti-GFAP antibody Polyclonal Rabbit WB, IF, IHC

 

Aging and Neurological Disorders

Product No. Description Clonality Species
Origin
Applications
(legend below)
HPA014784
Anti-AQP4
Polyclonal Rabbit WB, IHC
HPA021241 Anti-PHGDH Polyclonal Rabbit WB, IF, IHC
AMAB90905 Monoclonal anti-CD40 CL1673,
Monoclonal
Mouse WB, IHC
HPA010687 Anti-RHOT1 Polyclonal Rabbit WB, IHC

 

Development

Product No. Description Clonality Species
Origin
Applications
(legend below)
HPA000536
Anti-SOX11
Polyclonal Rabbit WB, IHC
HPA030775 Anti-PAX6 Polyclonal Rabbit WB, IF, IHC
HPA022040 Anti-RUNX2 Polyclonal Rabbit WB, IHC
HPA001762 ANTI-VIM Polyclonal Rabbit WB, IHC

 

FC = Flow Cytometry; ICC = Immunocytochemistry; IHC = Immunohistochemistry; IF = Immunofluorescence; IP = Immunoprecipitation; WB = Western Blot

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REPRODUCIBILITY> Added Consistency

Reproducibility is a bedrock principle and intrinsic challenge for researchers, authors and reviewers. For this reason, consistency found in everyday bioreagents is essential. Depend on our well characterized reagents such as buffers, carbohydrates and detergents to provide the stability you need to advance neuroscience.

CHALLENGE

Create consistent and reproducible results when detecting neural pathways.

SOLUTION

Consistency in core laboratory chemicals and products is required to ensure reproducibility when detecting neural pathways. For this reason we supply essential bioreagents with extensive specifications that are tested for suitability in multiple applications. Our added experience and knowledge in manufacturing biochemicals means we deliver lot-to-lot consistency, technical expertise and uninterrupted supply so you can reliably detect neural pathways.  

Learn more at sigmaaldrich.com/added-consistency