Confocal immunofluorescent analysis of U-251 MG cells (left, positive) and ZR-75-1 cells (right, negative) using Nestin (10C2) Mouse mAb (Alexa Fluor® 488 Conjugate) (green). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct immunofluorescent analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Nestin (10C2) Mouse mAb #33475.
|Immunofluorescence (Immunocytochemistry)||1:50 - 1:200|
Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised November 2013
Protocol Id: 182
Nestin (10C2) Mouse mAb (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of total nestin protein.
Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the carboxy terminus of human nestin protein.
Nestin is an intermediate filament family member protein that is structurally related to the neurofilament proteins (1). It is highly expressed in the developing brain, where it may help to regulate cell structure and intracellular processes required for neural cell division and migration (1,2). Upon maturation of the brain, nestin expression is quickly down-regulated and replaced by expression of the neurofilament proteins (2). Because nestin is expressed in both mature and precursor neuronal and glial cells, as well as in the developing brain and in the brain and spinal cord following damage, nestin is widely accepted as a marker of neural stem/progenitor cells (3). Research studies have shown that expression of nestin is also found in cells from various nervous system tumors, including gliomas, neuroblastomas, astrocytomas, and it is generally accepted as a marker for neural cancer stem cells (3). However, nestin expression has also been observed in astrocytes, retina, cardiac muscle, pancreas, and other tissues (3). Therefore, the acceptance of nestin as an exclusive marker of neural stem/progenitor cells is not unanimous.
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