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...

Manfred Andratsch, Norbert Mair, Cristina E. Constantin, Nadja Scherbakov, Camilla Benetti, Serena Quarta, Christian Vogl, Claudia A. Sailer, Nurcan Üceyler, Johannes Brockhaus, Rudolf Martini, Claudia Sommer, Hanns Ulrich Zeilhofer, Werner Müller, Rohini Kuner, John B. Davis, Stefan Rose-John, and Michaela Kress. A Key Role for gp130 Expressed on Peripheral Sensory Nerves in Pathological Pain. The Journal of Neuroscience, October 28, 2009, 29(43):13473-13483; doi:10.1523/JNEUROSCI.1822-09.2009

 ...DRGs were washed with PBS and fixed with 4% PFA for 20 min. After 3 x 10 min washing with PBS, ganglia were frozen in methylbutan. Sections (12 µm) were cut with a Leica CM 1850 Cryomicrotome, permeabilized with 0.1% TX-100, and blocked with blocking buffer. Sections were incubated with primary antibodies overnight at +4°C in a wet chamber. After washing in PBS, sections were incubated with the secondary antibodies, and embedded in Mowiol (Calbiochem). Indirect immune fluorescence was detected with a Zeiss Axiovert 200M microscope and analyzed with the MetaMorph Imaging Software Series 7.1 (Molecular Devices, Visitron Systems). The following antibodies were used: anti-gp130 (1:50, extracellular epitope, Neuromics); anti-Nav1.8 (1:200, was a generous gift from Prof. Dr. H. G. Knaus, Innsbruck, Austria) (see also supplemental Fig. 2, available at www.jneurosci.org as supplemental material) (Liu et al., 2006); anti-CGRP (1:300, ImmunoStar), anti-Isolectin IB4 (1:1000, Invitrogen); Alexa Fluor 488 goat anti-rabbit IgG (1:1000, H+L), Alexa Fluor 594, donkey anti-goat IgG (1:1000, all Invitrogen)...