Tuesday, October 21, 2014

Neurite Outgrowth Assays

Cells, Media and Markers

I have previously posted use of our Neurons in Live Content Assays for the study of Neurite Outgrowth: Neurons-Live Content Assays. These assays are critical for the study of repair and regeneration.

A recent publication featured several of our Neuron MarkersSerena Quarta, Bastian E. Baeumer, Nadja Scherbakov1, Manfred Andratsch, Stefan Rose-John, Georg Dechant3, Christine E. Bandtlow, and Michaela Kress: Peripheral Nerve Regeneration and NGF-Dependent Neurite Outgrowth of Adult Sensory Neurons Converge on STAT3 Phosphorylation Downstream of Neuropoietic Cytokine Receptor gp130. The Journal of Neuroscience, 24 September 2014, 34(39): 13222-13233; doi: 10.1523/JNEUROSCI.1209-13.2014.
Live labeling of neuron cultures: After 20 or 48 h, neurons were live-labeled with α-gp130 antibody diluted in cold TNB medium for 30 min on ice. After washing, neurons were incubated with the secondary antibody diluted in cold TNB medium for 30 min and washed with PBS. Cells were fixed either with 4% PFA for 20 min at room temperature (RT) or with methanol at −20°C for 2 min. After permeabilization with 0.01% TX-100 (Pierce) unspecific binding was blocked for 30 min with 10% normal goat serum (Sigma-Aldrich) in PBS. Cells were incubated with the first antibody for 1 h, washed three times for 10 min with PBS and incubated with the appropriate secondary antibody for 30 min, counterstained with 4′, 6-diamidino-2-phenylindole (1:10,000; Sigma-Aldrich) and embedded in Mowiol (Calbiochem). As primary antibodies, α-gp130 (1:50; Neuromics), α-β-III-tubulin clone TuJ-1 (1:1000; R&D Systems), and α-neurofilament-H (α-NF-H; 1:200; Neuromics) were used. Secondary antibodies used were α-goat AlexaFluor 594 (1:1000; Invitrogen), chicken α-mouse AlexaFluor 594 or donkey α-mouse AlexaFluor 488 (1:1000; Invitrogen), and goat α-chicken AlexaFluor 568 (1:10,000; Invitrogen) for fluorescence microcopy.

Images: Reduced density of TuJ-1+ nerve endings in the epidermis in SNS-gp130−/− mice after lesion. A, Representative cross sections of hindpaw glabrous skin of naive and 12 dpl gp130fl/fl and SNS-gp130−/− mice stained with the pan neuronal marker TuJ-1. The dotted line indicates the border between dermis and epidermis. Scale bar, 40 μm. B, Quantification of the total number of TuJ-1+ fibers (NE) per 1000 μm2 of epidermal area shows a significant decrease in density in SNS-gp130−/− mice after lesion compared with control animals (*p < 0.05; n = 4 for each group). Data are presented as mean ± SEM and analyzed by Mann–Whitney U test. C, 3D reconstruction of the deeper layer of the dermis shows fewer nerve bundles in SNS-gp130−/− dermis compared with controls. D, No NF-H+ proprioceptive fibers were detectable in the epidermis of gp130fl/fl animals at 12 dpl. Scale bar, 40 μm.

If you want to learn more about our Neuron-Glial-Astrocyte based assay solutions do not hesitate to contact me (612-801-1007) or pshuster@neuromics.com. Pete Shuster, Owner and CEO, Neuromics.

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