Immunofluorescence analysis of neurons

There are a wide variety of protein targets important when studying neurobiology. These targets are important in the study of glia, protein trafficking, neurogenesis, axon guidance, dendrite development, neuronal migration, growth factors, neuromuscular junction, neurotrophins, apoptosis, and synaptogenesis. We have a diverse array of highly specific antibodies for neuroscience research that have been validated* for multiple applications. These applications include western blot, flow cytometry, immunofluorescence, ELISA, and immunohistochemistry.

Markers relevant to neurobiology research

Neurobiology antibodies cover a wide range of targets and markers depending on the type of research being done. Listed below are some of the most prevalent markers that are studied. For a more in depth list, you can download the Invitrogen Neurobiology Antibodies poster.

  • Neurodegenerative disease research targets
    • Neurodegenerative research targets include cleavage specific antibodies to amyloid beta, phosphospecific, and pan antibodies against tau, parkin, LRRK2, nuclear aggregation of huntingtin (HTT) and MBP. Targeted neurodegenerative diseases include: Alzheimer’s disease, amyotrophic lateral sclerosis, Huntington’s disease, Multiple sclerosis, and Parkinson’s disease.
      Markers of interest: beta amyloid, C9orf72, HTT, IL-17A, alpha synuclein
  • Neuron markers
    • Neuron markers allow the specific identification of different neuronal cells in samples. Specific neuronal subtypes, like dopaminergic, glutamatergic and motor neurons express specific proteins that allow them to be specifically detected in imaging and flow cytometry experiments.
      Markers of interest: ChAT, OTX2, GAD65, NMDAR2B, ISL2, calretinin
  • Glial progenitor cell markers
    • Glial progenitor cells can be differentiated from neural stem cells, and then further differentiated into astrocytes and oliogodendrocytes. These progenitor cells are characterized by expression of CD44 and A2B5.
      Markers of interest: A2B5, CD44
  • Presynaptic and postsynaptic markers
    • Neuron communication involves the release of neurotransmitters from the presynaptic cells to the post synaptic cells. These cells express specific markers like synaptophysin (presynaptic) and PSD95 (postsynaptic) that allow for the differentiation of cell types.
      Markers of interest: HOMER1, PSD95, synaptophysin, synaptotagmin, VGLUT1
  • Oligodendrocyte progenitor cell and oligodendrocyte markers
    • Oligodendrocyte cells support and insulate neurons, creating the myelin sheath. They can be differentiated from glial progenitor cells. Important markers like GalC can be used to identify oligodendrocytes in a variety of applications and sample types.
      Markers of interest: NG2, OLIG1, OLIG2, PDGFRA, MBP
  • Neuroectoderm markers
    • Mammalian neurogenesis begins with the induction of neuroectoderm, which forms a neural plate and then folds to give rise to the neural tube. These structures are made up of a layer of neuroepithelial progenitors (NEPs).NEPs can be rapidly turned into primitive neural stem cells (NSCs).
      Markers of interest: NCAM, SOX2, OTX2, PAX3, PAX6
  • Neural stem cell markers
    • Neuronal stem cells are the first step in neuronal development from embryonic stem cells or induced pluripotent stem cells. The express important markers, nestin and SOX1 in addition to others. Neuronal stem cells can be further differentiated into glial progenitor cells and neuronal progenitor cells.
      Markers of interest: SOX1, SOX2, PAX3, PAX6, FOXG1
  • Astrocyte markers
    • Astrocytes cells support the neural network. They express neurotransmitter receptors and may possibly play a role in neuronal communication. They express the important marker GFAP and can be differentiated from glial progenitor cells.
      Markers of interest: ALDH1L1, EAAT2, GFAP, glutamine synthetase, S100beta
  • Microglia markers
    • Microglia play a major role in the immune system in the central nervous system, phagocytosing foreign material and cellular debris. Microglia can activate pro-inflammatory cytokines like, IFN-gamma, IL1-alpha, and TNF-alpha. Microglia also synthesize amyloid precursor protein (APP) in response to injury.
      Markers of interest: CD11b, CD68, CX3CR1, HLA-DR, IBA1

Applications

Our growing portfolio of traditional and recombinant antibodies is designed to enable detection and characterization of neurobiology targets with exceptional specificity.

Immunofluorescent analysis of FOXG1 in A549 cells

Immunofluorescent analysis of A549 cells using a FOXG1 polyclonal antibody (Cat. No. PA5-26794). A549 cells were fixed with 4% PFA (20 minutes), permeabilized with Triton X-100 (0.1%, 10 minutes), then incubated with a FOXG1 polyclonal antibody (Cat. No. PA5-26794) (1:25, 1 hour at 37°C). Primary antibody was detected with fluor-conjugated donkey anti-rabbit secondary antibody (green) at 1:400 dilution for 50 minutes at 37°C). Actin filaments have been labeled with dye-conjugated phalloidin (red). Nuclei were counterstained with DAPI (blue) (10 µg/mL, 10 minutes).

Chromatin immunoprecipitation analysis of Pax3. Performed using cross-linked chromatin from 1 x 10^6 HCT116 human colon carcinoma cells treated with serum for 0, 15, and 60 minutes. Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay with 1.0 µL/100 µL well volume of a Pax3 Recombinant Rabbit Polyclonal Antibody (Cat. No. 710178). Chromatin aliquots from ~1 x 10^5 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1 µL of eluted DNA in 2 µL SYBR real-time PCR reactions containing primers to amplify-3.2kb upstream of human FOS, exon-4 of FOS, or the imprinting control region (ICR) of the human H19 locus. PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean +/- SEM for three experiments. A schematic representation of human FOS and H19 loci are shown above the data where boxes represent exons (grey boxes = translated regions, white boxes = untranslated regions), the zigzag lines represent introns, and the straight lines represent upstream sequences. Regions amplified by FOS and H19 primers are represented by black bars.

ChIP analysis of PAX3 in human colon carcinoma cells
Western blot analysis of beta amyloid

Western blot analysis on membrane enriched extract (30 µg) of HEK 293 (Lane 1), SK-N-AS (Lane 2), SH-SY5Y (Lane 3) and U-87 MG (Lane 4). The blots were probed with Anti-beta-Amyloid Rabbit Polyclonal Antibody (Cat. No. 36-6900, 2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Cat. No. A27036, 0.4 µg/mL 1:2500 dilution). A ~ 35 kDa band corresponding to beta-Amyloid was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex NuPAGE 12% Bis-Tris gel (Cat. No. NP0342BOX), XCell SureLock Electrophoresis System (Cat. No. EI0002) and Novex Sharp Pre-Stained Protein Standard (Cat. No. LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with Pierce Power Blotter System (Cat. No. PB0012). The membrane was probed with the relevant primary and secondary Antibody using iBind Flex Western Starter Kit (Cat. No. SLF2000S). Chemiluminescent detection was performed using Pierce ECL Western Blotting Substrate (Cat. No. 32106).

*The use or any variation of the word “validation” refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses