Human/Mouse/Rat Neural 3-Color Immunocytochemistry Kit
R&D Systems, part of Bio-Techne | Catalog # SC024
Key Product Details
Summary for Human/Mouse/Rat Neural 3-Color Immunocytochemistry Kit
Key Benefits
- Verifies multipotency to reduce experimental variation
- Labels neurons, astrocytes, and oligodendrocytes
- Can be used for simultaneous 3-color staining
Why Analyze Neural Progenitor Cell Proliferation and Differentiation In Vitro?
Research studies using neural progenitor cells (NPCs) most often require weeks of cell culture before an experimental hypothesis can be tested.
Thus, the ability to verify multipotency at an early stage in the experimental process is an important time saving step. Multipotency status can be evaluated by the presence of cell markers and/or the ability of progenitor cells to differentiate into multiple neural lineages. To determine if a cell population truly consists of multipotent NPCs, it is important to verify their ability to differentiate into neurons, astrocytes, and oligodendrocytes.
3-color ICC using neural cell markers:
- Evaluates the multipotency status of the starting NPC population.
- Increases confidence in multipotency status by combining 3 different fluorochrome-conjugated antibodies for simultaneous staining.
- Reduces variability within an experiment and between studies.
- Ensures efficient use of time and reagents by utilizing single-step staining.
- Can be performed using randomly differentiated samples.
This kit contains the following fluorochrome-conjugated antibodies to verify neural stem cell multipotency:
- NL557-conjugated Mouse anti-Human Oligodendrocyte Marker O4
- NL637-conjugated Mouse anti-beta III Tubulin
- NL493-conjugated Sheep anti-Human GFAP
Each antibody is supplied as a 10X stock; enough for 25 assays when used in a 50 µL staining volume per assay.
Neural stem cells provide an excellent model for research focused on neural development and neurological disorders. R&D Systems offers ready-to-use primary cortical stem cells isolated from E14.5 Sprague-Dawley rats. In addition, primary mouse cortical stem cells isolated from E14.5 CD-1 mice are available. Every lot of R&D Systems Cortical Stem Cells is validated for a high level of Nestin expression and the capacity for multi-lineage differentiation into astrocytes, neurons, and oligodendrocytes. Our cortical stem cells are tested to ensure highest quality and lot to lot consistency. Both rat and mouse Cortical Stem Cells can be optimally expanded as monolayers or neurospheres.
To complement the use of primary neural stem cells, we offer a range of supportive products, including culture media which is specifically optimized for use with neural stem cells. We offer kits to promote the in vitro proliferation of neural precursors, and kits to differentiate neural stem cells into dopaminergic neurons or oligodendrocytes. In addition, kits are available which contain panels of antibodies designed to monitor the differentiation and identification of neural precursors, astrocytes, neurons, and oligodendrocytes.
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Beta-III Tubulin, GFAP, and Oligodendrocyte Marker O4 in Differentiated Rat Cortical Stem Cells. Beta-III Tubulin, GFAP, and Oligodendrocyte Marker O4 were simultaneously detected in immersion-fixed 7 day differentiated Rat Cortical Stem Cells (Catalog # NSC001) using the Human/Mouse/Rat Neural 3-Color Immunocytochemistry Kit (Catalog # SC024). Proteins were detected using antibodies included in the kit: neurons were stained with a NorthernLights™(NL)637-conjugated Mouse Anti-neuron Specific beta-III Tubulin Monoclonal Antibody (pseudocolored gray), astrocytes were stained with a NL493-conjugated Sheep Anti-GFAP Antigen Affinity-purified Polyclonal Antibody (green), and oligodendrocytes were stained with a NL557-conjugated Mouse Anti-Oligodendrocyte Marker O4 Monoclonal Antibody (red). The nuclei were counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Stem Cells on Coverslips. |
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, neural stem cell (NSC) multipotency can be verified via 3-Color ICC using the following protocol:
- NSCs are cultured on coated coverslips
- Fluorochrome-conjugated primary antibodies are added to fixed cells
- Neural cell markers are visualized using a fluorescence microscope
Reagents supplied in the Human/Mouse/Rat Neural 3-Color Immunocytochemistry Kit (Catalog # SC024):
- NL557-conjugated Mouse anti-Human Oligodendrocyte Marker O4
- NL637-conjugated Mouse anti-beta III Tubulin
- NL493-conjugated Sheep anti-Human GFAP
Reagents
- Appropriate NSC culture substrate (e.g., Human Fibronectin (Catalog # 1918-FN-02M), Bovine Fibronectin (Catalog # 1030-FN), Poly-L-ornithine, etc.)
- Cell culture medium (e.g., DMEM and N-2 Plus Media Supplement (Catalog # AR003) or equivalent)
- 4% paraformaldehyde in PBS
- 1% BSA in PBS
- 0.3% Triton™ X-100, 1% BSA, 10% normal donkey serum in PBS
- Mounting medium (Catalog # CTS011 or equivalent)
Materials
- Differentiated NPCs
- Coverslips (sterilized)
- Cell culture plate (24-well)
Equipment
- 37 °C and 5% CO2 incubator
- Centrifuge
- Hemocytometer
- Inverted microscope
- Fluorescence microplate
These antibodies have been tested for immunocytochemistry staining using 7 day differentiated rat cortical stem cells.
Coat coverslips with a NSC culture substrate.
Plate NSCs
Culture to desired confluency/age
Fix differentiated cells with 4% paraformaldehyde.
Block in blocking solution.
Incubate with fluorochrome-conjugated primary antibodies.
Wash with wash buffer.
Incubate with nuclear counterstain.
48 hours Mount the coverslip.
Incubate Visualize using a fluorescence microscope and appropriate filter sets.
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