Saturday, September 22, 2012

Neuron-specific class III beta-tubulin (TuJ1) Staining in hNT2.19 Neurons

Our Tuj-1 antibody is widely used and frequently published. It is proving a potent marker for confirming the differentiation of human neural progenitors to neurons.

In this study the authors use the marker for staining hNT2.19 Neurons: Mary J. Eaton, Yerko Berrocal, and Stacey Q. Wolfe. Potential for Cell-Transplant Therapy with Human Neuronal Precursors to Treat Neuropathic Pain in Models of PNS and CNS Injury: Comparison of hNT2.17 and hNT2.19 Cell Lines. Pain Research and Treatment. Volume 2012 (2012), Article ID 356412, 31 pages. doi:10.1155/2012/356412. 
The results show great promise. They show hNT2 or hNT2-derived cell lines, such as hNT2.17 and hNT2.19, have great potential to permanently reverse symptoms of neuropathic pain following PNS and CNS injuries and can offer new hope to treat these intractable conditions to significantly improve human health. This includes neuropathic pain resulting from diabetic neuropathy and Spinal Cord Injury.

Images: Comparison of graft sites of hNT2.17 and hNT2.19 in the QUIS and severe contusive-SCI models, respectively, at 6 weeks after cell transplant. (a) Sagittal section of anti-GABA-immunostained QUIS + hNT2.17 transplant lumbar spinal cord 6 weeks after grafting. Easily detectible hNT2.17 cells stain for GABA (arrows) on the pial membranes. (b) Sagittal section of anti-NuMA-immunostained QUIS + hNT2.17 transplant lumbar spinal cord 6 weeks after grafting. Easily detectible hNT2.17 cells stain for NuMA (arrows) on the pial membranes in adjacent sections. The hNT2.19 were alternately injected into the subarachnoid space two weeks after severe contusive SCI. Cell graft sites were co-localized with 5HT (c) and the human-specific marker TuJ1(d) (neuron-specific class III β-tubulin). There are many surviving hNT2.19 (d) grafted cells visible on the pial surface, which stain for TuJ1 (arrows) at the end of the experiment, 56 days after SCI and about 6 weeks after cell transplant. Adjacent sections with the same grafted hNT2.19 (c) are labeled for 5HT (arrows). Magnification bar = 20 μm.

Protocol: Modified methods for staining spinal cord sections for the human neuron-specific class III beta-tubulin (TuJ1) to identify grafted hNT2.19 neurons after grafting have previously been described [51]. The sections were washed with 0.1 M PBS pH 7.4 and permeabilized with 0.4% Triton-X-100 in 0.1 M PBS, 10% normal goat serum (NGS) for one hour. The sections were then incubated overnight at 4°C in the primary anti-TuJ1 antibody (1 : 100 DPBS), and the permeabilizing solution, followed by a one-hour incubation at room temperature with the secondary antibody solution, biotinylated mouse IgG raised in goat (Vector; 1 : 200), a Peroxidase ABC reporter in 0.1 M PBS (Vector) and “VIP” substrate (Vector). Some sections were stained in the absence of primary antibody and served as the negative controls.

I will continue to post new applications for our stem cell research reagents.

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