Yiting Liu, Katherine S. Given, Danielle E. Harlow, Adeline M. Matschulat, Wendy B. Macklin, Jeffrey L. Bennett and Gregory P. Owens. Myelin-specific multiple sclerosis antibodies cause complement-dependent oligodendrocyte loss and demyelination. Acta Neuropathologica Communications Neuroscience of Disease 20175:25 DOI: 10.1186/s40478-017-0428-6
After treatment, cerebellar slices were rinsed twice with PBS and fixed in 4% paraformaldehyde in PBS for 20 min at 4 °C. For immunohistochemistry, slices were rinsed in PBS and permeabilized in 1.5 or 10% (myelin proteins) Triton X-100 in PBS for 20 min. Slices were rinsed, blocked with 5% normal donkey serum (NDS) in PBS with 0.3% Triton X-100 for 1 h, and incubated with primary antibodies overnight at room temperature. Following 3 washes in PBS, Alexa Fluor-labeled secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were applied (1:800) overnight at room temperature, washed 3 times in PBS and mounted in Fluoromount G (Southern Biotech, Birmingham, AL). The following primary antibodies were used: rabbit anti-GFAP (Sigma), rabbit anti-Calbindin (Millipore), mouse anti-MAG (Millipore), rabbit anti-MOG (Abcam, Cambridge, United Kingdom), mouse anti-MBP (Covance, Princeton, NJ), chicken anti- Neurofilament-H (Neuromics, Minneapolis, MN), goat anti-Iba1 (Abcam), mouse anti-C3d (a gift from Dr. Joshua Thurman, University of Colorado), rabbit anti-MAC complex (anti C5b-9, Abcam), guinea pig anti- NG2 and rabbit anti-Olig2 (are gifts from Dr. Charles Stiles, Harvard University).
Prior to immunostaining, PFA-fixed mouse cerebellum tissue sections were thawed for 10 min, re-hydrated in PBS for 10 min, and blocked in PBS containing 3% bovine serum albumin (BSA), 2% normal goat serum (NGS), and 0.3% Triton-X100 for 1 h. MS rAbs were applied at a final concentration of 20 μg/mL to mouse tissues for 16 h at 4 °C in PBS containing 3% BSA and 2% NGS. Tissue sections were then washed 3 times for 3 min with PBS. Alexa fluorescent secondary antibody (1:1000) against human IgG (Molecular probes, Life Science Technologies) was applied for 1 h at room temperature in PBS containing 3% BSA and 2% NGS. Tissue was then washed 5 times for 3 min in PBS and mounted using Vectashield (Vector Laboratories, Burlingame, CA, USA) containing DAPI....
Rashad Hussain and Wendy B. Macklin. Integrin linked kinase (ILK) deletion disrupts oligodendrocyte development by altering cell cycle. Journal of Neuroscience 2 December 2016, 2113-16; DOI: http://dx.doi.org/10.1523/JNEUROSCI.2113-16.2016
...For neurons, mouse anti NeuN (1:1000, MAB 164 377, EMD MilliPore) and chicken antiYneurofilament (1:2000, CH22104, Neuromics); for 165 astrocytes, mouse antiYGFAP (1:600, G3898, Sigma). ...
...the neurons were fixed with 4% PFA for 30 min after 72 h in culture and were blocked with 10% goat serum, 1% BSA, 0.3% Triton X-100 in PBS for 30 min and then were incubated with a chicken anti-Neurofilament-medium (NF-M) antibody (1:1000, Neuromics, Edina, MN) and ...
Bethany L. Johnson-Kerner, Faizzan S. Ahmad, Alejandro Garcia Diaz, John Palmer Greene, Steven J. Gray, Richard Jude Samulski, Wendy K. Chung, Rudy Van Coster, Paul Maertens, Scott A. Noggle, Christopher E. Henderson, and Hynek Wichterle. Intermediate filament protein accumulation in motor neurons derived from giant axonal neuropathy iPSCs rescued by restoration of gigaxonin. Hum. Mol. Genet., 1 March 2015; 24: 1420 - 1431.
Jie Xu, Rodney J Nash, Teryl K Frey. Cellular responses to Sindbis virus infection of neural progenitors derived from human embryonic stem cells. BMC Research Notes 2014, 7:757 doi:10.1186/1756-0500-7-757.
...Standard immunocyto-fluorescence was performed. hNPCs grown on coated glass-coverslips (80% confluence) were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 10 minutes at room temperature, rinsed twice with PBS and then permeabilized with 0.2% TritonX-100 diluted in PBS for 5–7 min. Primary antibodies used were directed against Nestin (a neural stem marker; 1:450; Neuromics), SINV-NSP (a SINV antigen, 1:100,Eptomics), active-caspase 3 (1:500; Cell signaling), and β-tubulin (cytoskeleton marker, 1:200, Abcam). Secondary antibodies were anti-rabbit/mouse Alexa Fluor 488 and anti-rabbit/mouse Alexa Fluor 594 (1:2000–4000; Molecular Probes-Invitrogen Life Technologies). Fluorescence images were acquired on a Zeiss Axioplan epifluorescence wide-field microscope and processed with AxioVision software. For each condition within the same experiment, at least 3 fields were analyzed. For image quantification, at least three fields in the same experiment were analyzed...Western Blot analysis was performed on cell lysates of hNPCs, either mock infected or SINV infected, at 4, 12, 24, 36, and 48 hours post infection, using protocols described previously . Primary antibodies used were anti-GAPDH (1:5000; Abcam), anti-NF-kB p65 (1:200; Santa Cruz); anti-phospho-STAT3 (1:200; Cell signaling), anti-phospho-IRF3 (1:1000; Eptomics); anti-Nestin (1:500; Neuromics); anti-neuro-filament M (NF-M; 1:500; Neuromics); anti-Tuj1 (1:1000; Abcam); anti-PCNA (1:1000, Santa Cruz) and anti-cleavedcaspase 3 (1:1000; Cell Signaling). For quantification of western blot, films of immunoblot were scanned with a flat-bed scanner, and digital images were imported and quantified using Image J software . Then, the intensities of bands were compared according to their grayscale (http://www.lukemiller.org/journal/2007/08/quantifying-western-blotswithout.html
Bethany L. Johnson-Kerner, Faizzan S. Ahmad, Alejandro Garcia Diaz, J. Palmer Greene, Steven J. Gray, R. Jude Samulski, Wendy K. Chung, Rudy Van Coster, Paul Maertens, Scott A. Noggle, Christopher E. Henderson and Hynek Wichterle. Intermediate filament protein accumulation in motor neurons derived from giant axonal neuropathy iPSCs rescued by restoration of gigaxonin. Hum. Mol. Genet. (2014) doi: 10.1093/hmg/ddu556. First published online: November 4, 2014.
Serena Quarta, Bastian E. Baeumer, Nadja Scherbakov, Manfred Andratsch, Stefan Rose-John, Georg Dechant, 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
...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...
Kiran Vasudeva, Karl Andersen, Bree Zeyzus-Johns, T. Kevin Hitchens, Sravan Kumar Patel, Anthony Balducci, Jelena M. Janjic, John A. Pollock. Imaging Neuroinflammation In Vivo in a Neuropathic Pain Rat Model with Near-Infrared Fluorescence and F Magnetic Resonance. Published: February 28, 2014 DOI: 10.1371/journal.pone.0090589.
...1:50 mouse anti-rat Neurofilament-M (Neuromics, Edina, MN)...
Gayle M. Passmore, Joanne M. Reilly, Matthew Thakur, Vanessa N. Keasberry, Stephen J. Marsh, Anthony H. Dickenson and David A. Brown. Functional significance of M-type potassium channels in nociceptive cutaneous sensory endings. Fronteirs in Molecular Science. doi: 10.3389/fnmol.2012.00063.
Juan M Jimenez-Andrade and Patrick W Mantyh. Sensory and sympathetic nerve fibers undergo sprouting and neuroma formation in the painful arthritic joint of geriatric mice. Arthritis Research & Therapy 2012, 14:R101
...to label primary afferent sensory nerve fibers, an antibody against neurofilament 200 kDa (NF200, chicken anti neurofilament 200 kDa; NF200, 1:5000; Neuromics; catalog #CH22104)...
Marong Fang, Jing Wang, Jian-Ying Huang, Shu-Cai Ling, john A. Rudd, Zhi-Ying Hu, David T. Yew, Shu Han. The Neuroprotective Effects of Reg-2 Following Spinal Cord Transection Injury. The Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology. Volume 294, Issue 1, pages 24–45, January 2011.
...rabbit anti-160KD NF-M primary antibodies (1:1000, Neuromics)...
Juan M. Jimenez-Andrade, Aaron P. Bloom, James I. Stake, William G. Mantyh, Reid N. Taylor, Katie T. Freeman, Joseph R. Ghilardi, Michael A. Kuskowski, and Patrick W. Mantyh Pathological Sprouting of Adult Nociceptors in Chronic Prostate Cancer-Induced Bone Pain. J. Neurosci., Nov 2010; 30: 14649 - 14656 ; doi:10.1523/JNEUROSCI.3300-10.2010
...Sensory nerve fibers expressing the 200 kDa neurofilament (NF200) were labeled with an antibody against NF200 (anti-chicken, 1:5000; Neuromics, catalog # CH22104).
Agnès Gardet, Yair Benita, Chun Li, Bruce E. Sands, Isabel Ballester, Christine Stevens, Joshua R. Korzenik, John D. Rioux, Mark J. Daly, Ramnik J. Xavier, and Daniel K. Podolsky LRRK2 Is Involved in the IFN- Response and Host Response to Pathogens J. Immunol., Nov 2010; 185: 5577 - 5585
Min-Tsai Liu, Yung-Hui Kuan, Jingwen Wang, René Hen, and Michael D. Gershon. 5-HT4 Receptor-Mediated Neuroprotection and Neurogenesis in the Enteric Nervous System of Adult Mice. The Journal of Neuroscience, August 5, 2009, 29(31):9683-9699; doi:10.1523/JNEUROSCI.1145-09.2009.
Antibodies Refernced in this Publication::